Use of alkylated semi-synthetic glycosaminoglycosan ethers for the treatment of dental disorders

ABSTRACT

Described herein are methods for treating a dental disorder in a subject comprising administering to the subject an effective amount of a modified hyaluronan or a pharmaceutically acceptable salt or ester thereof, wherein said hyaluronan or its pharmaceutically acceptable salt or ester comprises at least one sulfate group and the primary C-6 hydroxyl proton of at least one N-acetyl-glucosamine residue is substituted with a C 1 -C 10  unsubstituted alkyl group or fluoroalkyl group.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 12/870,774, filed on Aug. 27, 2010, which is a continuation-in-partof International Application No. PCT/US09/039498, filed Apr. 3, 2009,which claims priority to U.S. provisional application Ser. No.61/042,310, filed Apr. 4, 2008. These applications are herebyincorporated by reference in their entirety for all of their teachings.

BACKGROUND

Inflammatory diseases such as psoriasis, dermatitis, acne, rosacea,photo-dermal ageing, and numerous diseases linked to RAGE-mediatedsignaling plague people worldwide. To put these diseases intoperspective, the National Psoriasis Foundation reports that psoriasisalone afflicts 2-3% of the world's population or approximately 125million people. These inflammatory conditions can be aestheticallyunpleasing and can create serious health issues if left untreated.

Conventionally accepted treatments of these conditions may involve UVphototherapy, corticosteroids and glucocorticoids, acitretin,cyclosporine, and methotrexate. However, each of these treatments maycause serious side effects ranging from immune suppression and liverdisease to thinning skin and causing birth defects. Due to partial orcomplete ineffectiveness, these treatments often leave patientsunsatisfied with their results.

In addition to the treatments mentioned above, heparin treatment hasalso been experimentally explored. Heparin, a sulfated polysaccharide,has traditionally been used almost exclusively as an anti-coagulant, butits anti-inflammatory properties are well known. Heparin and itsderivatives have shown some promise in treating these inflammatorydiseases. Particularly heparin and its derivatives disrupt at leastthree important events in inflammatory cascades. First, heparin attachesto and blocks the leukocyte integrins P- and L-selectin. Second, heparinand its derivatives reduce the inflammatory cascade by binding to andinhibiting the cationic PMN protease human leukocyte elastase andcathepsin G, which reduces proteolytic tissue injury by PMNs that escapethe first heparin barrier of selectin inhibition. Third, heparin and itsderivatives potentially inhibit the interaction of the receptor foradvanced glycation end-products (RAGE) with its ligands.

Although heparin and its derivatives have shown promise in treatingthese inflammatory diseases, treatment with heparin and its derivativesexhibits several major drawbacks. First, heparin and its derivatives areporcine-derived; thus leading to concerns of cross-species transfer ofviruses. Second, because of heparin's anticoagulant properties,diabetics treated with this compound are at risk of excessive bleeding.Third, heparin may induce thrombocytopenia in certain individuals whoproduce an antibody to the complex of heparin with the cationic proteinplatelet factor-4 (PF-4), resulting in catastrophic platelet aggregationand generalized paradoxical arterial and venous clotting. Thus, animportant unmet need is to formulate compounds which may be used totreat inflammatory diseases while avoiding the myriad of side effectsseen in other treatments.

SUMMARY

Described herein is the synthesis of alkylated and fluoroalkylatedsemi-synthetic glycosaminoglycosan ethers, referred to herein as“SAGEs.” The synthesis of sulfated alkylated and fluoroalkylated SAGEsis also described. The compounds described herein are useful in a numberof therapeutic and cosmetic applications and the treatment of a numberof inflammatory diseases and skin disorders. The advantages of theinvention will be set forth in part in the description which follows,and in part will be obvious from the description, or may be learned bypractice of the aspects described below. The advantages described belowwill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 shows a synthetic scheme for producing alkylated andfluoroalkylated hyaluronan and sulfated derivatives thereof.

FIG. 2 shows the structures of several exemplary SAGEs.

FIG. 3 shows the inhibition of P-Selectin by partially O-sulfated andmethylated HA (P-OSMEHA, or GM-131201).

FIG. 4 shows the inhibition of human leukocyte elastase by sulfatedhyaluronan derivatives, including alkylated and fluoroalkylatedhyaluronans, which are also sulfated.

FIG. 5 shows the inhibition of amphoterin, also known as high mobilitygroup box protein-1 (HMGB-1) binding to immobilized RAGE by P-OSMEHA.

FIG. 6 shows the inhibition of S100b calgranulin binding to immobilizedRAGE by P-OSMEHA (GM-131201).

FIG. 7 shows the inhibition of carboxymethyl lysine-BSA (CML-BSA)binding to immobilized RAGE by P-OSMEHA (GM-131201).

FIG. 8 shows the inhibition of keratinocyte proliferation by highmolecular weight hyaluronan derivatives, including alkylated andfluoroalkylated hyaluronans, that are also sulfated.

FIG. 9 shows the inhibition of keratinocyte proliferation by lowmolecular weight hyaluronan derivatives, including alkylated andfluoroalkylated hyaluronans, which are also sulfated.

FIG. 10 shows SAGE (GM-111101) co-injection with LL-37 rosacea model.FIG. 10A shows a gross picture of LL-37 injected skin region and FIG.10B shows co-injection model of LL-37 and SAGE. FIG. 10C showsH&E-stained cross-sectional view of a LL-37 injected skin sample. FIG.10D shows H&E-stained cross-sectional view of a LL-37 mixed with SAGEinjected skin region. FIG. 10E shows polymorphonuclear leukocyte (PMN)infiltration into skin as measured by activity of the PMN enzymemyeloperoxidase (MPO) in skin biopsies from miced injected with LL-37only, LL-37 plus SAGE or SAGE only injection groups. FIG. 10F shows thearea of erythema and FIG. 10G shows the erythema score in mice injectedwith LL-37 only or LL-37 plus SAGE.

FIG. 11 shows SAGE (GM-111101) topical treatment in the LL-37 rosaceamodel. FIG. 11A shows the gross picture of LL-37 injected skin region.FIG. 11B shows SAGE treatment immediately after LL-37 injection and FIG.11C shows SAGE treatment 12 h after LL-37 injection. FIG. 11D showsH&E-stained cross-sectional view of a LL-37 injected skin sample. FIG.11E shows H&E-stained cross-sectional view of SAGE immediate treatmentin LL-37 injected skin region. FIG. 11F shows H&E-stainedcross-sectional view of SAGE 12 h treatment in LL-37 injected skinregion. FIG. 11G shows MPO activity measurement of LL-37 injection modelwith different SAGE treatment strategies. FIG. 11H shows the area oferythema and FIG. 11I shows the erythema score demonstration of LL-37rosacea model treated with topical application of SAGE.

FIG. 12A shows the outer Skin under natural light (treated with 1 mg/mlSAGE) and outer Skin fluorescent image (FIG. 12B). FIG. 12C shows theinner skin under natural light and FIG. 12D shows the inner skin underfluorescent condition.

FIG. 13A shows the effects of HA derivatives on the proliferation ofnHDF cells and effect on nHEK (FIG. 13B). Gross pictures of mice treatedwith different concentrations of GM-111101 and GM-212101. The intactarea (FIG. 13C) and formic acid irritated area (FIG. 13D) were comparedwith 0.1 mg/ml GM-111101 (FIG. 13E), 1 mg/ml GM-111101 (FIG. 13F), 10mg/ml GM-111101 (FIG. 13G), 0.1 mg/ml GM-212101 (FIG. 13H), 1 mg/mlGM-212101 (FIG. 13I) and 10 mg/ml GM-212101 (FIG. 13J).

FIG. 14A shows erythema scoring of SAGE in abraded area. FIG. 14B showsedema scoring of SAGE in abraded area. FIG. 14C shows erythema scoringof SAGE in intact area. FIG. 14D shows edema scoring of SAGE in intactarea.

FIG. 15 shows SAGE treatment using croton oil inflammatory model. Fourhours after croton oil treatment in control (CTL) group. Comparisonbetween Right (untreated) (FIG. 15A) and Left (croton oil painted) (FIG.15B) ears of the same mouse in CTL group. H&E staining was done fornegative control with PBS painting (FIG. 15C), positive control withcroton oil (FIG. 15D) and croton oil followed by SAGE treatment (FIG.15E). Leukocytic infiltration and edema was identified in the croton oilpositive control group. Myeloperoxidsae activity (FIG. 15F) is an indexof polymorphonuclear leukocyte activation and was measured in earpunches after SAGE treatment. FIG. 15G and FIG. 15H show changes in earthickness (from edema) and ear redness (from irritation) after SAGEtreatment. (p<0.05)

FIG. 16A shows H&E-stained cross-sectional view of a LL-37 injected skinsample. FIG. 16A shows H&E-stained cross-sectional view of HA treatmentin LL-37 injected skin region. FIG. 16C shows H&E-stainedcross-sectional view of SAGE (GM-111101) treatment in LL-37 injectedskin region. FIG. 16D shows MPO activity measurement of LL-37 injectionmodel with HA and SAGE treatment. FIG. 16E shows the area of erythemaillustration and FIG. 16F shows the erythema score demonstration ofLL-37 rosacea model with HA and SAGE treatment.

FIG. 17 shows AGE-induced RAGE expression in ARPE-19 cells is increasedby growth on the AGE product carboxymethyl lysine-bovine serum albumin(CML-BSA) and prevented by modified heparin.

FIG. 18A shows that compared to control, the AGE product CML-BSA inducesapoptosis in ARPE-19 cells (FIG. 18B) that is inhibited by ODSH (FIG.18C) and almost eliminated by SAGE treatment (FIG. 18D).

FIG. 19 shows that LMW SAGEs do not activate Factor XII, unlike heparinwhich activates Factor XII at a concentration of 0.4 μg/ml, which isclose to the therapeutic anticoagulating plasma heparin concentration inhumans.

DETAILED DESCRIPTION

Before the present compounds, compositions, and/or methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific compounds, synthetic methods, or uses assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a pharmaceutical carrier” includes mixtures of two or moresuch carriers, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted lower alkyl”means that the lower alkyl group can or can not be substituted and thatthe description includes both unsubstituted lower alkyl and lower alkylwhere there is substitution.

References in the specification and concluding claims to parts byweight, of a particular element or component in a composition orarticle, denotes the weight relationship between the element orcomponent and any other elements or components in the composition orarticle for which a part by weight is expressed. Thus, in a compoundcontaining 2 parts by weight of component X and 5 parts by weightcomponent Y, X and Y are present at a weight ratio of 2:5, and arepresent in such ratio regardless of whether additional components arecontained in the compound.

A weight percent of a component, unless specifically stated to thecontrary, is based on the total weight of the formulation or compositionin which the component is included.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. For example, hyaluronanthat contains at least one —OH group can be represented by the formulaY—OH, where Y is the remainder (i.e., residue) of the hyaluronanmolecule.

The term “treat” as used herein is defined as maintaining or reducingthe symptoms of a pre-existing condition. The term “prevent” as usedherein is defined as eliminating or reducing the likelihood of theoccurrence of one or more symptoms of a disease or disorder. The term“inhibit” as used herein is the ability of the compounds describedherein to completely eliminate the activity or reduce the activity whencompared to the same activity in the absence of the compound.

Described herein are alkylated and fluoroalkylated hyaluronan orderivatives thereof. In one aspect, at least one primary C-6 hydroxylproton of the N-acetyl-glucosamine residue of hyaluronan is substitutedwith an alkyl group. The term “alkyl group” as used herein is a branchedor unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, suchas methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl,tetracosyl and the like. In one aspect, the alkyl group is a C₁-C₁₀branched or straight chain alkyl group. In a further aspect, the alkylgroup is methyl. The alkyl group can be unsubstituted or substituted. Inthe case when the alkyl group is substituted, one or more hydrogen atomspresent on the alkyl group can be replaced with or more groupsincluding, but not limited to, alkynyl, alkenyl, aryl, halide, nitro,amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, aralkyl, oralkoxy.

In another aspect, at least one primary C-6 hydroxyl proton of theN-acetyl-glucosamine residue of hyaluronan is substituted with afluoroalkyl group. The term “fluoroalkyl group” as used herein is abranched or unbranched saturated hydrocarbon group of 1 to 24 carbonatoms, wherein at least one of the hydrogen atoms is substituted withfluorine. In certain aspects, the fluoroalkyl group includes at leastone trifluoromethyl group. In other aspects, the fluoroalkyl group hasthe formula —CH₂(CF₂)_(n)CF₃, wherein n is an integer of 1, 2, 3, 4, 5,6, 7, 8, 9, or 10. In one aspect, the fluoroalkyl group is —CH₂CF₂CF₃ or—CH₂CF₂CF₂CF₃.

Described herein are methods for alkylating or fluoroalkylating SAGEs.In one aspect, the SAGEs are produced by (a) reacting the hyaluronan ora derivative thereof with a sufficient amount of base to deprotonate atleast one primary C-6 hydroxyl proton of the N-acetyl-glucosamineresidue, and (b) reacting the deprotonated hyaluronan or a derivativethereof with an alkylating agent or fluoroalkylating for a sufficienttime and concentration to alkylate or fluoroalkylate at least onedeprotonated primary C-6 hydroxyl group. It will be understood by thoseskilled in the art that the basic conditions may also lead to cleavageof the glycosidic linkage, leading to lower molecular weight hyaluronanderivatives during the modification process. It will also be understoodthat the basic conditions deprotonate the acid to the carboxylate, andthe secondary hydroxyl groups, and that each of these nucleophilicmoieties may participate in the ensuing alkylation in proportion totheir relative abundance at equilibrium and the nucleophilicity of theanionic species. For example, 2-O and/or 3-O hydroxyl protons can bedeprotonated and alkylated or fluoroalkylated. An example of this isdepicted in FIG. 1, where R can be hydrogen, an alkyl group, or an alkylgroup.

The hyaluronan starting material can exist as the free acid or the saltthereof. Derivatives of hyaluronan starting material can also be usedherein. The derivatives include any modification of the hyaluronan priorto the alkylation or fluoroalkylation step. A wide variety of molecularweight hyaluronan can be used herein. In one aspect, the hyaluronan hasa molecular weight greater than 10 kDa prior to alkylation orfluoroalkylation. In another aspect, the hyaluronan has a molecularweight from 25 kDa to 1,000 kDa, 100 kDa to 1,000 kDa, 25 kDa to 500kDa, 25 kDa to 250 kDa, or 25 kDa to 100 kDa prior to alkylation orfluoroalkylation. In certain aspects, the hyaluronan starting materialor a derivative thereof is not derived from an animal source. In theseaspects, the hyaluronan can be derived from other sources such asbacteria. For example, a recombinant B. subtilis expression system canbe used to produce the hyaluronan starting material.

The hyaluronan starting material or derivative thereof is initiallyreacted with a sufficient amount of base to deprotonate at least oneprimary C-6 hydroxyl proton of the N-acetyl-glucosamine residue. Theselection of the base can vary. For example, an alkali hydroxide such assodium hydroxide or potassium hydroxide can be used herein. Theconcentration or amount of base can vary depending upon the desireddegree of alkylation or fluoroalkylation. In one aspect, the amount ofbase is sufficient to deprotonate at least 0.001% of the primary C-6hydroxyl protons of the N-acetyl-glucosamine residue of the hyaluronanstarting material or derivative thereof. In other aspects, the amount ofbase is sufficient to deprotonate from 0.001% to 50%, 1% to 50% 5% to45%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 50%, 20% to 50%, or 30% to50% of the primary C-6 hydroxyl protons of the N-acetyl-glucosamineresidue of the hyaluronan starting material or derivative thereof. It isunderstood that the more basic the solution, the more likely are chaincleavage reactions and the higher the degree ofalkylation/fluoroalkylation that can be achieved. For example, otherhydroxyl groups present on hyaluronan (e.g., 2-OH and/or 3-OH can bealkylated or fluoroalkylated). In one aspect, all of the hydroxyl groupspresent on hyaluronan can be alkylated or fluoroalkylated. In otheraspects, 0.001%, 0.01%, 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 100%, or any range thereof of hydroxyl protons present onhyaluronan can be deprotonated and subsequently alkylated orfluoroalkylated.

After the hyaluronan starting material or derivative thereof has beentreated with a base, the deprotonated hyaluronan is reacted with analkylating agent or fluoroalkylating agent to produce the SAGE. Examplesof alkylating agents include, but are not limited to, an alkyl halide.Alkyl bromides and iodides are particularly useful. Similarly, thefluoroalkylating agent can include a fluoroalkyl halide. Alkylatingagents and fluoroalkylating agents commonly used in organic synthesiscan be used herein.

An exemplary synthetic procedure for making alkylated andfluoroalkylated SAGEs is provided in FIG. 1. Referring to FIG. 1,hyaluronan (HA) is treated with a base (e.g., NaOH) and an alkylatingagent (e.g., CH₃I) to methylate a primary C-6 hydroxyl proton of theN-acetyl-glucosamine residue of hyaluronan and produce methylatedhyaluronan (MHA). FIG. 1 also provides an exemplary synthetic procedurefor making a fluoroalkylated hyaluronan (FHA) using a fluoroalkylatingagent (e.g., CF₃(CF₂)_(n)CH₂Br).

In certain aspects, it is desirable to sulfate the alkylated orfluoroalkylated SAGEs described above. In one aspect, the alkylated orfluoroalkylated SAGE is sulfated by reacting the alkylated orfluoroalkylated SAGE with a sulfating agent to produce a sulfatedproduct. The degree of sulfation can vary from partial sulfation tocomplete sulfation. In general, free hydroxyl groups present on thealkylated or fluoroalkylated hyaluronan or a derivative thereof can besulfated. In one aspect, at least one C-2 hydroxyl proton and/or C-3hydroxyl proton is substituted with a sulfate group. In another aspect,the degree of sulfation is from 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 or anyrange thereof per disaccharide unit of the alkylated or fluoroalkylatedSAGE. In one aspect, the alkylated or fluoroalkylated SAGE can betreated with a base to deprotonate one or more hydroxyl protons followedby the addition of the sulfating agent. The sulfating agent is anycompound that reacts with a hydroxyl group or deprotonated hydroxylgroup to produce a sulfate group. The molecular weight of the SAGE canvary depending upon reaction conditions. In one aspect, the molecularweight of the SAGE is from 2 kDa to 500 kDa, 2 kDa to 250 kDa, 2 kDa to100 kDa, 2 kDa to 50 kDa, 2 kDa to 25 kDa, or from 2 kDa to 10 kDa. FIG.1 depicts an exemplary synthesis of sulfated alkylated orfluoroalkylated SAGEs (SMHA and SFHA, respectively).

FIG. 2 provides the structures of several exemplary SAGEs. Each SAGE isidentified by the code is GM-XYSTZZ, where:

X=type of alkyl group, where 1=methyl, 2=pentafluoropropyl,3=heptafluorobutyl, 4=benzylglycidyl ether

Y=size of HA, where 1=low, 2=medium, 3=high

S=degree of sulfation, where 1=partial, 2=full

T=degree of alkylation, where 1=low, 2=high

ZZ=sequential lot number 01 or 02, where the 02 has been made and hasall the same properties as the 01 batch.

Table 1 provides a list of several SAGEs as defined by the code systemabove.

TABLE 1 Alkylation SAGE # CHEMICAL NAME MW(starting) MW(GPC) AlkylationSD Sulfation GM-211101 LMW-P-OSFHA-1(DS 1)  53K  6K Pentafluoropropyl(Pfp) 1 1.0-1.5 GM-311101 LMW-P-OSFHA-2(DS 1)  53K  5.8KHeptafluorobutyl (Hfb) 1 1.0-1.5 GM-111101 LMW-P-OSMeHA(DS 1)  53K  5.6kMethyl (Me) 1 1.0-1.5 GM-211201 LMW-P-OSFHA-1(DS 2)  53K  6Kpentafluoropropyl 2 1.0-1.5 GM-311201 LMW-P-OSFHA-2(DS 2)  53K  5.6kheptafluorobutyl 2 1.0-1.5 GM-111201 LMW-P-OSMeHA(DS 2)  53K  5.5Kmethyl 2 1.0-1.5 GM-231101 P-OSFHA-1(DS 1) 950K 112k Pentafluoropropyl 11.0-1.5 GM-331101 P-OSFHA-2(DS 1) 950K 110k Heptafluorobutyl (Hfb) 11.0-1.5 GM-131101 P-OSMeHA(DS 1) 950K 123k methyl 1 1.0-1.5 GM-231201P-OSFHA-1(DS 2) 950K 108k pentafluoropropyl 2 1.0-1.5 GM-331201P-OSFHA-2(DS 2) 950K 130k heptafluorobutyl 2 1.0-1.5 GM-131201P-OSMeHA(DS 2) 950K 120K methyl 2 1.0-1.5 GM-212101 LMW-F-OSFHA-1(DS 1) 53K  5k pentafluoropropyl 1 1.5-2.0 GM-312101 LMW-F-OSFHA-2(DS 1)  53K 4.8k heptafluorobutyl 1 1.5-2.0 GM-112101 LMW-F-OSMeHA(DS 1)  53K  5.6kmethyl 1 1.5-2.0 GM-212201 LMW-F-OSFHA-1(DS 2)  53K  6Kpentafluoropropyl 2 1.5-2.0 GM-312201 LMW-F-OSFHA-2(DS 2)  53K  6Kheptafluorobutyl 2 1.5-2.0 GM-112201 LMW-F-OSMeHA(DS 2)  53K  5.4kmethyl 2 1.5-2.0 GM-232101 F-OSFHA-1(DS 1) 950K 110k pentafluoropropyl 11.5-2.0 GM-332101 F-OSFHA-2(DS 1) 950K 105k heptafluorobutyl 1 1.5-2.0GM-132101 F-OSMeHA(DS 1) 950K 112k Methyl 1 1.5-2.0 GM-232201F-OSFHA-1(DS 2) 950K 120k pentafluoropropyl 2 1.5-2.0 GM-332201F-OSFHA-2(DS 2) 950K 118k heptafluorobutyl 2 1.5-2.0 GM-132201F-OSMeHA(DS 2) 950K 116K methyl 2 1.5-2.0 GM-431101 P-OSBGHA 950K 105kbenzyl glycidyl ether <1 (BG) GM-432101 F-OSBGHA 950K 110k benzylglycidyl ether <1 GM-411101 P-OSBGHA  53K  6K benzyl glycidyl ether <1GM-412101 F-OSBGHA  53K  5.6k benzyl glycidyl ether <1 GM-XYSTZZ codingX = alkyl 1 = Me 2 = Pfp 3 = Hfb 4 = BG Y = MW 1 = Low 2 = Medium 3 =High S = sulfation 1 = partial 2 = full T = Alkylation 1 = Low SD 2 =high SD ZZ = Sequential no.

In one aspect, the alkyl group of the SAGE is methyl and at least oneC-2 hydroxyl proton and/or C-3 hydroxyl proton of hyaluronan issubstituted with a sulfate group. In another aspect, the alkyl group ofthe SAGE is methyl, at least one C-2 hydroxyl proton and/or C-3 hydroxylproton of hyaluronan is substituted with a sulfate group, and thecompound has a molecular weight of 2 kDa to 200 kDa after alkylation. Anexample of such a compound is GM-111101 as shown in FIG. 2.

Any of the alkylated and fluoroalkylated SAGEs described herein can bethe pharmaceutically acceptable salt or ester thereof. Pharmaceuticallyacceptable salts are prepared by treating the free acid with anappropriate amount of a pharmaceutically acceptable base. Representativepharmaceutically acceptable bases are ammonium hydroxide, sodiumhydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide,magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copperhydroxide, aluminum hydroxide, ferric hydroxide, isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine,ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine,arginine, histidine, and the like. In one aspect, the reaction isconducted in water, alone or in combination with an inert,water-miscible organic solvent, at a temperature of from about 0° C. toabout 100° C. such as at room temperature. The molar ratio of compoundsof structural formula I to base used are chosen to provide the ratiodesired for any particular salts. For preparing, for example, theammonium salts of the free acid starting material, the starting materialcan be treated with approximately one equivalent of pharmaceuticallyacceptable base to yield a neutral salt.

Ester derivatives are typically prepared as precursors to the acid formof the compounds—as illustrated in the examples below—and accordinglycan serve as prodrugs. Generally, these derivatives will be lower alkylesters such as methyl, ethyl, and the like. Amide derivatives —(CO)NH₂,—(CO)NHR and —(CO)NR₂, where R is an alkyl group defined above, can beprepared by reaction of the carboxylic acid-containing compound withammonia or a substituted amine. Also, the esters can be fatty acidesters. For example, the palmitic ester has been prepared and can beused as an alternative esterase-activated prodrug.

The SAGEs described herein can be formulated in any excipient thebiological system or entity can tolerate to produce pharmaceuticalcompositions. Examples of such excipients include, but are not limitedto, water, aqueous hyaluronic acid, saline, Ringer's solution, dextrosesolution, Hank's solution, and other aqueous physiologically balancedsalt solutions. Nonaqueous vehicles, such as fixed oils, vegetable oilssuch as olive oil and sesame oil, triglycerides, propylene glycol,polyethylene glycol, and injectable organic esters such as ethyl oleatecan also be used. Other useful formulations include suspensionscontaining viscosity enhancing agents, such as sodiumcarboxymethylcellulose, sorbitol, or dextran. Excipients can alsocontain minor amounts of additives, such as substances that enhanceisotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.In certain aspects, the pH can be modified depending upon the mode ofadministration. For example, the pH of the composition is from about 5to about 6, which is suitable for topical applications. Additionally,the pharmaceutical compositions can include carriers, thickeners,diluents, preservatives, surface active agents and the like in additionto the compounds described herein.

The pharmaceutical compositions can also include one or more activeingredients used in combination with the compounds described herein. Theresulting pharmaceutical composition can provide a system for sustained,continuous delivery of drugs and other biologically-active agents totissues adjacent to or distant from the application site. Thebiologically-active agent is capable of providing a local or systemicbiological, physiological or therapeutic effect in the biological systemto which it is applied. For example, the agent can act to control and/orprevent infection or inflammation, enhance cell growth and tissueregeneration, control tumor growth, act as an analgesic, promoteanti-cell attachment, reduce alveolar bone and tooth loss, inhibitdegeneration of cartilage and weight bearing joints, and enhance bonegrowth, among other functions. Additionally, any of the compoundsdescribed herein can contain combinations of two or morepharmaceutically-acceptable compounds. Examples of such compoundsinclude, but are not limited to, antimicrobial agents, antiinflammatoryagents, anesthetics, and the like. Methods for using these compositionsas drug delivery devices is described in detail below.

The pharmaceutical compositions can be prepared using techniques knownin the art. In one aspect, the composition is prepared by admixing aSAGE described herein with a pharmaceutically-acceptable compound and/orcarrier. The term “admixing” is defined as mixing the two componentstogether so that there is no chemical reaction or physical interaction.The term “admixing” also includes the chemical reaction or physicalinteraction between the compound and the pharmaceutically-acceptablecompound. Covalent bonding to reactive therapeutic drugs, e.g., thosehaving nucleophilic groups, can be undertaken on the compound. Second,non-covalent entrapment of a pharmacologically active agent in across-linked polysaccharide is also possible. Third, electrostatic orhydrophobic interactions can facilitate retention of apharmaceutically-acceptable compound in the compounds described herein.

It will be appreciated that the actual preferred amounts of SAGE in aspecified case will vary according to the specific compound beingutilized, the particular compositions formulated, the mode ofapplication, and the particular situs and subject being treated. Dosagesfor a given host can be determined using conventional considerations,e.g. by customary comparison of the differential activities of thesubject compounds and of a known agent, e.g., by means of an appropriateconventional pharmacological protocol. Physicians and formulators,skilled in the art of determining doses of pharmaceutical compounds,will have no problems determining dose according to standardrecommendations (Physicians Desk Reference, Barnhart Publishing (1999).

The pharmaceutical compositions described herein can be administered ina number of ways depending on whether local or systemic treatment isdesired, and on the area to be treated. Administration can be topically(including ophthalmically, vaginally, rectally, intranasally, orally, ordirectly to the skin). Formulations for topical administration caninclude ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like can be necessary ordesirable. Administration can also be directly into the lung byinhalation of an aerosol or dry micronized powder. Administration canalso be by direct injection into the inflammed or degenerating jointspace.

Preparations for administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous carriersinclude water, alcoholic/aqueous solutions, emulsions or suspensions,including saline and buffered media. Parenteral vehicles, if needed forcollateral use of the disclosed compositions and methods, include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's, or fixed oils. Intravenous vehicles, if needed forcollateral use of the disclosed compositions and methods, include fluidand nutrient replenishers, electrolyte replenishers (such as those basedon Ringer's dextrose), and the like. Preservatives and other additivescan also be present such as, for example, antimicrobials, anti-oxidants,chelating agents, and inert gases and the like.

Dosing is dependent on severity and responsiveness of the condition tobe treated, but will normally be one or more doses per day, with courseof treatment lasting from several days to several months or until one ofordinary skill in the art determines the delivery should cease. Personsof ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates.

The SAGEs and pharmaceutical compositions described herein can be usedin a variety of applications related to drug delivery, small moleculedelivery, wound healing, treatment of inflammatory skin disorders,treatment of inflammatory dental disorders, treatment of inflammatoryrespiratory disorders, treatment of inflammatory eye disorders, burninjury healing, and tissue regeneration/engineering. In one aspect, theSAGEs and compositions described herein can improve wound healing in asubject in need of such improvement. The SAGEs and pharmaceuticalcompositions described herein can be placed directly in or on anybiological system without purification as it is composed ofbiocompatible materials. Examples of sites the SAGEs can be placedinclude, but not limited to, soft tissue such as muscle or fat; hardtissue such as bone or cartilage; areas of tissue regeneration; a voidspace such as periodontal pocket; surgical incision or other formedpocket or cavity; a natural cavity such as the oral, vaginal, rectal ornasal cavities, the joint space, the cul-de-sac of the eye, and thelike; the peritoneal cavity and organs contained within, and other sitesinto or onto which the compounds can be placed including a skin surfacedefect such as a cut, scrape or burn area. It is contemplated that thetissue can be damaged due to injury or a degenerative condition or, inthe alternative, the SAGEs and compositions described herein can beapplied to undamaged tissue to prevent injury to the tissue. The SAGEscan be biodegradable and naturally occurring enzymes will act to degradethem over time. Components of the SAGE can be “bioabsorbable” in thatthe components of the SAGE will be broken down and absorbed within thebiological system, for example, by a cell, tissue and the like.Additionally, SAGEs, especially those that have not been rehydrated, canbe applied to a biological system to absorb fluid from an area ofinterest.

In the case of wound healing, the SAGEs described herein can beadministered via injection. For many clinical uses, when the SAGE is inthe form of a hydrogel film, injectable hydrogels are preferred forthree main reasons. First, an injectable hydrogel could be formed intoany desired shape at the site of injury. Because the initial hydrogelscan be sols or moldable putties, the systems can be positioned incomplex shapes and then subsequently crosslinked to conform to therequired dimensions. Second, the hydrogel would adhere to the tissueduring gel formation, and the resulting mechanical interlocking arisingfrom surface microroughness would strengthen the tissue-hydrogelinterface. Third, introduction of an in situ-crosslinkable hydrogelcould be accomplished using needle or by laparoscopic methods, therebyminimizing the invasiveness of the surgical technique.

In the case of inflammatory skin disorders such as psoriasis, acne,atopic dermatitis, rosacea or UV light dependent photo-aging, the SAGEscan be applied topically as part of an emollient to prevent or treat theintended condition. In the case of respiratory disorders such as asthma,chronic obstructive pulmonary disease, acute lung injury or cysticfibrosis, the SAGEs can be dissolved in a water-soluble isotonic vehiclecompatible with airway lining fluid and delivered to the lung or nasalpassages as an inhaled aerosol. Alternately, the SAGEs can be formulatedinto a micronized powder and inhaled into the lung as a dry powder. Inthe case of eye diseases, the SAGEs can be placed into an aqueousvehicle and applied to the eye topically as drops, or injected directlyinto the eye either by needle or using an implanted constant drugdelivery device. In the case of dental disorders such as periodontaldisease, the SAGEs can be added as a component of a mouthwash orformulated into creams or gingival packing materials to be applieddirectly to the gingival crevice.

The SAGEs can also be injected parenterally either intravenously,intramuscularly or subcutaneously to treat or prevent systemicinflammatory disorders such as diabetic vascular or renal disease orinflammatory gastrointestinal diseases. Similarly, the SAGEs can beinjected intra-articularly to treat inflammatory and degenerativearthritis. The SAGEs can also be administered orally in capsules orformulated into an enema to be delivered intra-rectally as treatment forinflammatory bowel diseases.

The SAGEs and compositions described herein can deliver at least onepharmaceutically-acceptable compound to a patient in need of suchdelivery, comprising contacting at least one tissue capable of receivingthe pharmaceutically-acceptable compound with one or more compositionsdescribed herein. The SAGEs can be used as a carrier for a wide varietyof releasable biologically active substances having curative ortherapeutic value for human or non-human animals. Many of thesesubstances that can be carried by the SAGE are discussed above. Includedamong biologically active materials which are suitable for incorporationinto the gels of the invention are therapeutic drugs, e.g.,anti-inflammatory agents, anti-pyretic agents, steroidal andnon-steroidal drugs for anti-inflammatory use, hormones, growth factors,contraceptive agents, antivirals, antibacterials, antifungals,analgesics, hypnotics, sedatives, tranquilizers, anti-convulsants,muscle relaxants, local anesthetics, antispasmodics, antiulcer drugs,peptidic agonists, sympathiomimetic agents, cardiovascular agents,antitumor agents, oligonucleotides and their analogues and so forth. Abiologically active substance is added in pharmaceutically activeamounts.

In one aspect, the sulfated alkylated and fluoroalkylated SAGEsdescribed herein can inhibit the activity of the receptor for AdvancedGlycation Endproducts (RAGE), P-selectin, or human leukocyte elastase.RAGE is highly expressed in human skin, where it is present on dermalfibroblasts, dendritic cells, keratinocytes, endothelial cells andmonocytes. RAGE is upregulated in sun-exposed skin by Advanced GlycationEnd-Products (AGE) and by the cytokine tumor necrosis factor-α. RAGEplays a prominent role in UV-induced photo-ageing, where its ligation byAGE products such as UV-induced carboxymethyl lysine (CML) promotes skinaging through stimulation of extracellular matrix production by dermalfibroblasts. The role of RAGE is likely to be even more prominent inpsoriasis because this disease is critically dependent on activatedT-lymphocytes for initiation of inflammation. T-lymphocytes may also bemechanistically important in acne and atopic dermatitis. In the case ofacne, elevated dermal levels of CD3⁺ and CD4⁺ T lymphocytes andmacrophages stimulate hyper-proliferation of keratinocytes in the ductsof follicles, producing the plugged follicular ducts that lead toformation of the acne comedone. In the case of atopic dermatitis, dermalantigens activate T_(H)2 lymphocytes which secrete cytokines such asinterleukin-4 (IL-4) and interleukin-13 (IL-13), resulting in therecruitment of eosinophils into skin. Eosinophils, in turn, releasecationic toxins such as major basic protein, which produces allergicskin disease. Thus, the potent RAGE inhibiting activity of the compoundsdescribed herein makes them useful in treating a variety of skindisorders including, but not limited to, acne, eczema, atopicdermatitis, psoriasis, or photo-dermal ageing.

In the adult state, RAGE is not always so entirely helpful to theorganism. Malignant tumors secrete amphoterin (or high mobility boxgroup protein-1, HMGB-1) as an autocrine factor and use the interactionof amphoterin with RAGE to promote primary tumor growth and metastasis.Blocking RAGE with a recombinant decoy (soluble RAGE or s-RAGE) reducestumor growth and inhibits metastasis. During sepsis, monocytes andmacrophages secrete amphoterin which interacts with RAGE on bloodvessels and other inflammatory cells to enhance the severity ofbacterial shock. Blocking this interaction with antibodies against RAGEprevents organ damage in severe sepsis. In the adult state, RAGE alsofunctions as a vascular adhesion receptor promoting the recruitment ofPMNs, monocytes and lymphocytes into areas of inflammation. BlockingRAGE blunts inflammatory cell influx. This has been previouslydemonstrated in animal models of multiple sclerosis, where competitiveblockade of vascular endothelial RAGE with s-RAGE prevents the influx ofactivated encephalitogenic T-lymphocytes into the central nervoussystem, and retards onset and progression of neurologic inflammation anddegeneration.

RAGE also interacts with a family of calcium binding proteins calledS100 calgranulins, which are secreted by PMNs, monocytes and lymphocytesas potent inflammation-promoting factors. Elevated levels of S100calgranulins are a prominent marker of PMN inflammation in acute lunginjury and in the airway secretions of patients with cystic fibrosis. Inthe eye, the interaction of S100 calgranulins with RAGE plays aprominent role leading to blindness in age-related macular degeneration.RAGE also binds the Alzheimer's β-amyloid peptide and the β sheets ofamyloid proteins. Through RAGE-related induction of neural cell deathand inflammation, RAGE-β sheet fibrillar interactions mediateAlzheimer's dementia and organ damage in systemic amyloidosis.

RAGE is also prominent in diabetes mellitus. When blood glucose iselevated, the aldehyde group of glucose randomly attaches to the aminesof cellular proteins, covalent adducts. In the presence of oxidants suchas hypochlorous acid (HOCl), the oxidant produced by PMNs, this glucosemoiety can then become oxidized. These oxidized, glycosylated proteinsare known as Advanced Glycation End-Products, (AGE). AGEs also bind theRAGE receptor avidly, and trigger RAGE-mediated signaling. AGE-RAGEsignaling accounts for the vascular endothelial dysfunction, poor woundhealing and accelerated arterial atherosclerosis characteristic ofpoorly controlled diabetes. In the eye, AGE-RAGE signaling produces theproliferation of retinal microvessels that leads to diabetic retinopathyand blindness. In the kidney, AGE-RAGE signaling accounts for theinitial renal hypertrophy and then fibrosis that causes diabetic renalfailure (diabetic nephropathy). AGE-RAGE signaling likewise producesapoptosis of endothelium, inhibits blood vessel growth and retardshealing of cutaneous diabetic ulcers.

The ability of the SAGEs to block RAGE makes them particularly valuableas therapeutic agents for inflammation. RAGE functions in utero as areceptor binding the growth promoting nuclear protein amphoterin, orhigh mobility box protein-1 (HMGB-1). There, the amphoterin-RAGEinteraction triggers growth signaling important for nervous systemdevelopment. In the adult state, RAGE is expressed in the cells ofvessel walls, neural tissues, cardiac myocytes, monocytes andmacrophages, T-lymphocytes, renal mesangial cells, and in skinfibroblasts, dendrocytes and keratinocytes. Thus, in one aspect, theSAGEs and compositions described herein can be used to safely reduce orprevent inflammation in a subject produced by a variety of differentmaladies attributed to RAGE-related diseases including, but not limitedto, cancer, multiple sclerosis, osteoarthritis, cystic fibrosis, sicklecell anemia, a cardiovascular inflammatory disorder, or a cardiovascularinflammatory disorder, or diabetic complications.

In other aspects, the SAGEs and compositions, as negatively chargedentities, can also be administered to bind and inhibit cationic skinpeptides derived from cathelicidins, thereby treating or preventing skindisorders. For example, the skin condition known as acne rosacea, whichis known to occur from excess skin expression of active cathelicidinpeptides, can be treated or prevented using the SAGEs described herein.Examples of skin disorders that can be treated or prevented using theSAGEs include, but are not limited to, rosacea, atopic dermatitis(eczema), allergic contact dermatitis, psoriasis, dermatitisherpetiformis, acne, diabetic skin ulcers and other diabetic wounds,burns (including relieving pain of thermal burns), sunburn (includingrelieving pain of sunburn), prevention of scarring after plasticsurgery, actinic keratoses, inflammation from insect bites, poison ivy,radiation-induced dermatitis/burn, facilitation of skin healing,prevention and treatment of keloid scarring, or the treatment ofseborrheic dermatitis.

Due to the ability of the SAGEs to inhibit RAGE activity and otherbiological mechanisms, the SAGEs have numerous therapeutic applicationsin addition to treating or preventing skin disorders. In one aspect, theSAGEs can be used in dental and oral surgery to treat gingivitis(periodontal disease) and aphthous ulcers. In other aspects, the SAGEscan be used in ophthalmological applications such as, for example, inthe treatment of age-related macular degeneration, diabetic retinopathy,dry eye syndrome and other inflammatory conjunctivitis, iritis, uveitis,allergic conjunctivitis, anti-inflammatory aid in cataract surgery, orin the prevention of corneal inflammation and scarring.

In further aspects, the SAGEs can be used in genitourinary applications(e.g., prevention of urinary tract infection, treatment of thetransitional cell cancer of the bladder and uroepithelial system;treatment of interstitial cystitis; and use as a vaginallubricant/protective to prevent transmission of sexually transmitteddiseases).

In another aspect, the SAGEs can be used to treat a number ofrespiratory disorders including cystic fibrosis, bronchiectasis,rhinitis (both allergic and perennial), sinusitis, emphysema and chronicbronchitis (COPD), acute lung injury/adult respiratory distresssyndrome, interstitial lung fibrosis, SARS, asthma, and respiratorysyncytial virus. In other aspects, the SAGEs can prevent and treatsnoring and obstructive sleep apnea, prevent infection by commonrespiratory pathogens (Stretococcus pneumoniae, Hemophilus influenzae,Staphylococcus, Mycoplasma pneumoniae, Chlamydial pneumonia, Gramnegative enteric infections) in immune suppressed hosts such as subjectswho are HIV positive or who have hematopoietic malignancies, or preventand treat otitis media.

The SAGEs can be used in cardiovascular applications (e.g., treating orpreventing acute coronary syndrome or atherosclerosis);hematological/oncological applications (e.g., prevention and treatmentof sickle cell anemia; prevention and treatment of metastatic disease;and prevention of hypercoagulable state of malignancy (Trousseau'ssyndrome)); treatment of infectious diseases (e.g., cerebral vascularocclusive syndromes and nephritis in Falciparum malaria, Yellow fever,Denge fever, systemic sepsis, and adjunctive treatment of HIV to preventviral fusion with and infection of target cells); treatment ofgastrointestinal diseases (e.g., ulcerative colitis, Crohn's disease ofthe bowel, Hemorrhoids, and the Prevention of stress ulceration of thestomach and esophagus); treatment of rheumatological and immunologicaldiseases (e.g., prevention and treatment of osteoarthritis, rheumatoidarthritis, systemic lupus erythematosis, prevention and treatment ofangioneurotic edema, Sjogren's syndrome, systemic sclerosis, systemicamyloidosis, and systemic mastocytosis); renal diseases (e.g.,prevention and treatment of diabetic nephropathy andglomerulonephritis); and neurologic diseases (e.g., multiple sclerosisand Alzheimer's dementia).

The SAGEs and compositions described herein are safer than other relatedtherapies. For example, heparin and other sulfated polysaccharides canreduce diabetic complications in both animal and clinical studies, andare particularly effective against diabetic nephropathy. However,heparins cannot be used in general clinical settings to prevent diabeticcomplications because the anticoagulant properties present an excessiverisk of bleeding. The SAGEs and compositions described herein possesslow anticoagulant activity, which is an important consideration forlong-term treatment, which is demonstrated below in the Examples.Additionally, the SAGEs have little to no toxicity, which is alsodemonstrated in the Examples.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions. Compounds described below areidentified by code numbers as defined above and referred to in FIG. 2.

I. Synthesis of Alkylated HA Derivatives

A. Preparation of Methyl HA (DS-2)

Hyaluronic acid (HA, Novozymes Biopolymers, 950 kDa) (2.0 g) wasdissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixturestirred for 2 h at room temperature (rt). The resulting viscous liquidwas transferred to a 400 mL beaker that contained 100 mL of isopropanol,and mixing was continued. To stirred mixture was added 10 mL ofiodomethane, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude methylated HA product. Thiscrude MeHA was dissolved in 250 mL of distilled water, the solution wasadjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed MeHA product was lyophilized to afford 1.25 g ofmethyl HA as a cottony mesh. ¹H NMR (D₂O, δ): 1.85 (s, 3H, NCH₃),3.20-3.80 (m, 10H, OCH+OCH₃). The substitution degree (SD) wasdetermined by ¹H NMR, SD=[(integration of methyl HA atδ3.20-3.8)−(integration of HA at δ3.20-3.8)]/(integration of NCH₃ at1.85), and was estimated to be SD=2, or an average of 2 methyl groupsper disaccharide unit. This suggests that both the primary hydroxyl andat least one secondary hydroxyl group were modified by this process.

B. Preparation of Methyl HA (DS-1)

Hyaluronic acid (HA, Novozymes Biopolymers, 950 kDa) (2.0 g) wasdissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixturestirred for 2 h at room temperature (rt). The resulting viscous liquidwas transferred to a 400 mL beaker that contained 100 mL of isopropanol,and mixing was continued. To stirred mixture was added 4 mL ofiodomethane, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude methylated HA product. Thiscrude MeHA was dissolved in 250 mL of distilled water, the solution wasadjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed MeHA product was lyophilized to afford 1.25 g ofmethyl HA as a cottony mesh. ¹H NMR (D₂O, δ): 1.85 (s, 3H, NCH₃),3.20-3.80 (m, 10H, OCH+OCH₃). The substitution degree (SD) wasdetermined by ¹H NMR, SD=[(integration of methyl HA atδ3.20-3.8)−(integration of HA at δ3.20-3.8)]/(integration of NCH₃ at1.85), and was estimated to be SD=1, or an average of 1 methyl groupsper disaccharide unit. This suggests that the primary hydroxyl and atleast one secondary hydroxyl group were modified by this process.

C. Preparation of FHA-2 (DS-1)

To a 25 mL flask containing 1.0 g of 2,2,3,3,4,4-heptafluoro-1-butanol(5 mmol), 1.3 mL of phosphorus tribromide (7.5 mmol) was added slowly.The mixture was stirred at 60° C. for 30 minutes, and then saturatedsodium bicarbonate solution (15 mL) was slowly added to quench thereaction. The aqueous solution was extracted with three 15-mL portionsof dichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beakerand the mixture stirred for 2 h at room temperature (rt). The resultingviscous liquid was transferred to a 400 mL beaker that contained 100 mLof isopropanol, and mixing was continued. To the stirred mixture wasadded the crude heptafluorobutyl bromide in 10 mL of isopropanol, andthe mixture was stirred for 24 h at rt. The resulting suspension wasfiltered to collect the crude FHA-2 product. This crude FHA-2 wasdissolved in 250 mL of distilled water, the solution was adjusted to pH˜7.0, and the solution was dialyzed against distilled water for 24 h,changing the external water bath four times during this period. Thedialyzed FHA-2 product was lyophilized to afford 1.5 g of FHA-2,designed as FHA-2 as cottony mesh. ¹H NMR (D₂O, δ): 1.82 (s, 3H, NCH₃),3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ): −115.3, −120.8. Thesubstitution degree (SD) was determined by ¹H NMR as 1.0.SD=[(integration of FHA-2 at δ3.15-3.80)−(integration of HA atδ3.20-3.8)]/[(integration of NCH₃ at 1.82)×(⅔)].

D. Preparation of FHA-2 (DS-2)

To a 25 mL flask containing 3.0 g of 2,2,3,3,4,4-heptafluoro-1-butanol(15 mmol), 2.5. mL of phosphorus tribromide (16 mmol) was added slowly.The mixture was stirred at 60° C. for 30 minutes, and then saturatedsodium bicarbonate solution (15 mL) was slowly added to quench thereaction. The aqueous solution was extracted with three 15-mL portionsof dichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beakerand the mixture stirred for 2 h at room temperature (rt). The resultingviscous liquid was transferred to a 400 mL beaker that contained 100 mLof isopropanol, and mixing was continued. To the stirred mixture wasadded the crude heptafluorobutyl bromide in 10 mL of isopropanol, andthe mixture was stirred for 24 h at rt. The resulting suspension wasfiltered to collect the crude FHA-2 product. This crude FHA-2 wasdissolved in 250 mL of distilled water, the solution was adjusted to pH˜7.0, and the solution was dialyzed against distilled water for 24 h,changing the external water bath four times during this period. Thedialyzed FHA-2 product was lyophilized to afford 1.5 g of FHA-2,designed as FHA-2 as cottony mesh. ¹H NMR (D₂O, δ): 1.82 (s, 3H, NCH₃),3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ): −115.3, −120.8. Thesubstitution degree (SD) was determined by ¹H NMR as 2.0.SD=[(integration of FHA-2 at δ3.15-3.80)−(integration of HA atδ3.20-3.8)]/[(integration of NCH₃ at 1.82)×(⅔)].

E. Preparation of FHA-1 (DS-1)

To a 25 mL flask containing 1.0 g of 2,2,3,3,-pentafluoro-1-propanol (5mmol), 1.3 mL of phosphorus tribromide (7.5 mmol) was added slowly. Themixture was stirred at 60° C. for 30 minutes, and then saturated sodiumbicarbonate solution (15 mL) was slowly added to quench the reaction.The aqueous solution was extracted with three 15-mL portions ofdichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beakerand the mixture stirred for 2 h at room temperature (rt). The resultingviscous liquid was transferred to a 400 mL beaker that contained 100 mLof isopropanol, and mixing was continued. To the stirred mixture wasadded the crude heptafluorobutyl bromide in 10 mL of isopropanol, andthe mixture was stirred for 24 h at rt. The resulting suspension wasfiltered to collect the crude FHA-1 product. This crude FHA-1 wasdissolved in 250 mL of distilled water, the solution was adjusted to pH˜7.0, and the solution was dialyzed against distilled water for 24 h,changing the external water bath four times during this period. Thedialyzed FHA-1 product was lyophilized to afford 1.5 g of FHA-1,designed as FHA-1 as cottony mesh. ¹H NMR (D₂O, δ): 1.80 (s, 3H, NCH₃),3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ): −113.6, −118.0. Thesubstitution degree (SD) was determined by ¹H NMR as 1.0.SD=[(integration of FHA-1 at δ3.15-3.80)−(integration of HA atδ3.20-3.8)]/[(integration of NCH₃ at 1.80)×(⅔)].

F. Preparation of FHA-1 (DS-2)

To a 25 mL flask containing 3.0 g of 2,2,3,3,-pentafluoro-1-propanol (15mmol), 3 mL of phosphorus tribromide (18 mmol) was added slowly. Themixture was stirred at 60° C. for 30 minutes, and then saturated sodiumbicarbonate solution (15 mL) was slowly added to quench the reaction.The aqueous solution was extracted with three 15-mL portions ofdichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mL beakerand the mixture stirred for 2 h at room temperature (rt). The resultingviscous liquid was transferred to a 400 mL beaker that contained 100 mLof isopropanol, and mixing was continued. To the stirred mixture wasadded the crude heptafluorobutyl bromide in 10 mL of isopropanol, andthe mixture was stirred for 24 h at rt. The resulting suspension wasfiltered to collect the crude FHA-1 product. This crude FHA-1 wasdissolved in 250 mL of distilled water, the solution was adjusted to pH˜7.0, and the solution was dialyzed against distilled water for 24 h,changing the external water bath four times during this period. Thedialyzed FHA-1 product was lyophilized to afford 1.5 g of FHA-1,designed as FHA-1 as cottony mesh. ¹H NMR (D₂O, δ): 1.80 (s, 3H, NCH₃),3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ): −113.6, −118.0. Thesubstitution degree (SD) was determined by ¹H NMR as 2.0.SD=[(integration of FHA-1 at δ3.15-3.80)−(integration of HA atδ3.20-3.8)]/[(integration of NCH₃ at 1.80)×(⅔)].

G. Preparation of BGHA

Hyaluronic acid (HA, Novozymes Biopolymers, 950 kDa) (2.0 g) wasdissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixturestirred for 2 h at room temperature (rt). The resulting viscous liquidwas transferred to a 400 mL beaker that contained 100 mL of isopropanol,and mixing was continued. To stirred mixture was added 10 mL of BenzylGlycidyl Ether, and the mixture was stirred for 24 h at rt. Theresulting suspension was filtered to collect the crude BGHA product.This crude BGHA was dissolved in 250 mL of distilled water, the solutionwas adjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed BGHA product was lyophilized to afford 1.25 g ofBGHA as a cottony mesh. ¹H NMR (D₂O, δ): 1.85 (s, 3H, NCH₃), 3.20-3.80(m, 10H, OCH+OCH₃). The substitution degree (SD) was determined by ¹HNMR, SD is less than 1.

H. Preparation of Low Molecular Weight Methyl HA (DS-2)

Hyaluronic acid (HA, Novozymes Biopolymers, 53 kDa) (2.0 g) wasdissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixturestirred for 2 h at room temperature (rt). The resulting viscous liquidwas transferred to a 400 mL beaker that contained 100 mL of isopropanol,and mixing was continued. To stirred mixture was added 10 mL ofiodomethane, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude Low molecule methylated HAproduct. This crude LMW MeHA was dissolved in 250 mL of distilled water,the solution was adjusted to pH ˜7.0, and the solution was dialyzedagainst distilled water for 24 h, changing the external water bath fourtimes during this period. The dialyzed LMW MeHA product was lyophilizedto afford 1.2 g of methyl HA as a cottony mesh. ¹H NMR (D₂O, δ): 1.85(s, 3H, NCH₃), 3.20-3.80 (m, 10H, OCH+OCH₃). The substitution degree(SD) was determined by ¹H NMR, as 2.

I. Preparation of Low Molecular Weight Methyl HA (DS-1)

Hyaluronic acid (HA, Novozymes Biopolymers, 53 kDa) (2.0 g) wasdissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixturestirred for 2 h at room temperature (rt). The resulting viscous liquidwas transferred to a 400 mL beaker that contained 100 mL of isopropanol,and mixing was continued. To stirred mixture was added 4 mL ofiodomethane, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude low molecule methylated HAproduct. This crude LMW MeHA was dissolved in 250 mL of distilled water,the solution was adjusted to pH ˜7.0, and the solution was dialyzedagainst distilled water for 24 h, changing the external water bath fourtimes during this period. The dialyzed LMW MeHA product was lyophilizedto afford 1.2 g of LMW MeHA as a cottony mesh. ¹H NMR (D₂O, δ): 1.85 (s,3H, NCH₃), 3.20-3.80 (m, 10H, OCH+OCH₃). The substitution degree (SD)was determined by ¹H NMR as 1.

J. Preparation of Low Molecular Weight FHA-2 (DS-1)

To a 25 mL flask containing 1.0 g of 2,2,3,3,4,4-heptafluoro-1-butanol(5 mmol), 1.3 mL of phosphorus tribromide (7.5 mmol) was added slowly.The mixture was stirred at 60° C. for 30 minutes, and then saturatedsodium bicarbonate solution (15 mL) was slowly added to quench thereaction. The aqueous solution was extracted with three 15-mL portionsof dichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g, 53 kDa was dissolved in 20 mL of NaOH (40% w/v) in a 100 mLbeaker and the mixture stirred for 2 h at room temperature (rt). Theresulting viscous liquid was transferred to a 400 mL beaker thatcontained 100 mL of isopropanol, and mixing was continued. To thestirred mixture was added the crude heptafluorobutyl bromide in 10 mL ofisopropanol, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude LMW FHA-2 product. Thiscrude LMW FHA-2 was dissolved in 250 mL of distilled water, the solutionwas adjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed LMW FHA-2 product was lyophilized to afford 1.5 gof LMW FHA-2, designed as LMW FHA-2 as cottony mesh. ¹H NMR (D₂O, δ):1.82 (s, 3H, NCH₃), 3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ):−115.3, −120.8. The substitution degree (SD) was determined by ¹H NMR as1.0.

K. Preparation of Low Molecular Weight FHA-2 (DS-2)

To a 25 mL flask containing 3.0 g of 2,2,3,3,4,4-heptafluoro-1-butanol(15 mmol), 2.5. mL of phosphorus tribromide (16 mmol) was added slowly.The mixture was stirred at 60° C. for 30 minutes, and then saturatedsodium bicarbonate solution (15 mL) was slowly added to quench thereaction. The aqueous solution was extracted with three 15-mL portionsof dichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g, 53 kDa) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mLbeaker and the mixture stirred for 2 h at room temperature (rt). Theresulting viscous liquid was transferred to a 400 mL beaker thatcontained 100 mL of isopropanol, and mixing was continued. To thestirred mixture was added the crude heptafluorobutyl bromide in 10 mL ofisopropanol, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude LMW FHA-2 product. Thiscrude LMW FHA-2 was dissolved in 250 mL of distilled water, the solutionwas adjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed LMW FHA-2 product was lyophilized to afford 1.5 gof LMW FHA-2, designed as LMW FHA-2 as cottony mesh. ¹H NMR (D₂O, δ):1.82 (s, 3H, NCH₃), 3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ):−115.3, −120.8. The substitution degree (SD) was determined by ¹H NMR as2.0.

L. Preparation of Low Molecular Weight FHA-1 (DS-1)

To a 25 mL flask containing 1.0 g of 2,2,3,3,-pentafluoro-1-propanol (5mmol), 1.3 mL of phosphorus tribromide (7.5 mmol) was added slowly. Themixture was stirred at 60° C. for 30 minutes, and then saturated sodiumbicarbonate solution (15 mL) was slowly added to quench the reaction.The aqueous solution was extracted with three 15-mL portions ofdichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g, 53 kDa) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mLbeaker and the mixture stirred for 2 h at room temperature (rt). Theresulting viscous liquid was transferred to a 400 mL beaker thatcontained 100 mL of isopropanol, and mixing was continued. To thestirred mixture was added the crude heptafluorobutyl bromide in 10 mL ofisopropanol, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude LMW FHA-1 product. Thiscrude LMW FHA-1 was dissolved in 250 mL of distilled water, the solutionwas adjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed LMW FHA-1 product was lyophilized to afford 1.5 gof LMW FHA-1, designed as LMW FHA-1 as cottony mesh. ¹H NMR (D₂O, δ):1.80 (s, 3H, NCH₃), 3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ):−113.6, −118.0. The substitution degree (SD) was determined by ¹H NMR as1.0.

M. Preparation of LMW FHA-1 (DS-2)

To a 25 mL flask containing 3.0 g of 2,2,3,3,-pentafluoro-1-propanol (15mmol), 3 mL of phosphorus tribromide (18 mmol) was added slowly. Themixture was stirred at 60° C. for 30 minutes, and then saturated sodiumbicarbonate solution (15 mL) was slowly added to quench the reaction.The aqueous solution was extracted with three 15-mL portions ofdichloromethane, the organic layer containing the heptafluorobutylbromide was concentrated, and the residue was used without purificationin the next step.

HA (2.0 g, 53 kDa) was dissolved in 20 mL of NaOH (40% w/v) in a 100 mLbeaker and the mixture stirred for 2 h at room temperature (rt). Theresulting viscous liquid was transferred to a 400 mL beaker thatcontained 100 mL of isopropanol, and mixing was continued. To thestirred mixture was added the crude heptafluorobutyl bromide in 10 mL ofisopropanol, and the mixture was stirred for 24 h at rt. The resultingsuspension was filtered to collect the crude LMW FHA-1 product. Thiscrude LMW FHA-1 was dissolved in 250 mL of distilled water, the solutionwas adjusted to pH ˜7.0, and the solution was dialyzed against distilledwater for 24 h, changing the external water bath four times during thisperiod. The dialyzed LMW FHA-1 product was lyophilized to afford 1.5 gof LMW FHA-1, designed as LMW FHA-1 as cottony mesh. ¹H NMR (D₂O, δ):1.80 (s, 3H, NCH₃), 3.15-3.80 (m, 8H, OCH+OCH₂). ¹⁹F NMR (D₂O, δ):−113.6, −118.0. The substitution degree (SD) was determined by ¹H NMR as2.0.

N. Preparation of Low Molecular Weight BGHA

Hyaluronic acid (HA, Novozymes Biopolymers, 53 kDa) (2.0 g) wasdissolved in 20 mL of NaOH (40% w/v) in a 100 mL beaker and the mixturestirred for 2 h at room temperature (rt). The resulting viscous liquidwas transferred to a 400 mL beaker that contained 100 mL of isopropanol,and mixing was continued. To stirred mixture was added 10 mL of BenzylGlycidyl Ether, and the mixture was stirred for 24 h at rt. Theresulting suspension was filtered to collect the crude LMW BGHA product.This crude LMW BGHA was dissolved in 250 mL of distilled water, thesolution was adjusted to pH ˜7.0, and the solution was dialyzed againstdistilled water for 24 h, changing the external water bath four timesduring this period. The dialyzed LMW BGHA product was lyophilized toafford 1.25 g of LMW BGHA as a cottony mesh. ¹H NMR (D₂O, δ): 1.85 (s,3H, NCH₃), 3.20-3.80 (m, 10H, OCH+OCH₃). The substitution degree (SD)was determined by ¹H NMR, SD is less than 1.

II. Sulfation of alkylated HA derivatives

1. Preparation of LMW-P-OSFHA-1 (DS-1) (GM-211101)

First, the tributylammonium (TBA) salt of LMW FHA-1 (DS-1) was preparedby the addition of 1 mL of tributylamine to LMW FHA-1 (1.0 g) in 100 mLof deionized water which was adjusted to pH 3.0 with 1N HCl. The mixturewas mixed vigorously, dried by lyophilization. The resulting salt(FHA-1-TBA) was dissolved in 25 mL of DMF to which the required excess(6 mol/equiv of available hydroxy group in HA) of pyridine-sulfurtrioxide complex (0.4 g) was added. After stirring for 3 h at 40° C.,the reaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated HA was dissolved in distilled waterand dialyzed against 100 mM of NaCl solution for two days, changing thesolution four times a day, and lyophilized to give the product (330 mg)in 75% yield and characterized by ¹H NMR, sulfation SD=1.0. Thesubstitution degree is determined by comparing the NMR shift of OCH tothose in literature (Carbohydrate Research, 1998, 306, 35-43).

2. Preparation of LMW-P-OSFHA-2 (DS-1) (GM-311101)

The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt (LMWFHA-2-TBA) was dissolved in 25 mL of DMF to which the required excess (6mol/equiv of available hydroxy group in HA) of pyridine-sulfur trioxidecomplex (0.4 g) was added. After stirring for 3 h at 40° C., thereaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated LMW FHA-2 was dissolved in water,dialyzed, and lyophilized as in 1. to give the product (300 mg) in 68%yield and characterized by ¹H NMR, sulfation SD=1.0.

3. Preparation of LMW-P-OSMeHA (DS-1) (GM-111101)

The TBA salt of LMW MeHA (DS-1) (from MW 53 kDa HA) was prepared from0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water as in 1.The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL of DMF to whichthe required excess (6 mol/equiv of available hydroxy groups in MeHA) ofpyridine-sulfur trioxide complex (0.8 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (540 mg)in 62% yield, which was shown by ¹H NMR to have a sulfation SD=1.0-1.5.

4. Preparation of LMW-P-OSFHA-1 (DS-2) (GM-211201)

The TBA salt of LMW FHA-1 (FHA-1 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt(FHA-1-TBA) was dissolved in 25 mL of DMF to which the required excess(6 mol/equiv of available hydroxy group in HA) of pyridine-sulfurtrioxide complex (0.4 g) was added. After stiffing for 3 h at 40° C.,the reaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated HA was dissolved in distilled waterand dialyzed against 100 mM of NaCl solution for two days, changing thesolution four times a day, and lyophilized to give the product (300 mg)in 70% yield and characterized by ¹H NMR, sulfation SD=1.0.

5. Preparation of LMW-P-OSFHA-2 (DS-2) (GM-311201)

The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt (LMWFHA-2-TBA) was dissolved in 25 mL of DMF to which the required excess (6mol/equiv of available hydroxy group in HA) of pyridine-sulfur trioxidecomplex (0.4 g) was added. After stirring for 3 h at 40° C., thereaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated LMW FHA-2 was dissolved in water,dialyzed, and lyophilized as in 1. to give the product (310 mg) in 70%yield and characterized by ¹H NMR, sulfation SD=1.0.

6. Preparation of LMW-P-OSMeHA (DS-2) (GM-111201)

The TBA salt of LMW MeHA (DS-1) (from MW 53 kDa HA) was prepared from0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water as in 1.The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL of DMF to whichthe required excess (6 mol/equiv of available hydroxy groups in MeHA) ofpyridine-sulfur trioxide complex (0.8 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (560 mg)in 64% yield, which was shown by ¹H NMR to have a sulfation SD=1.0-1.5.

7. Preparation of P-OSFHA-1 (DS-1) (GM-231101)

The TBA salt of FHA-1 (FHA-1 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL of distilledwater and processing as above in 1 The resulting salt (FHA-1-TBA) wasdissolved in 25 mL of DMF to which the required excess (6 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.4g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated HA was dissolved in distilled water and dialyzedagainst 100 mM of NaCl solution for two days, changing the solution fourtimes a day, and lyophilized to give the product (300 mg) in 68% yieldand characterized by ¹H NMR, sulfation SD=1.0-1.5.

8. Preparation of P-OSFHA-2 (DS-1) (GM-331101)

The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of distilledwater and processing as above in 1. The resulting salt (FHA-2-TBA) wasdissolved in 25 mL of DMF to which the required excess (6 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.4g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated FHA-2 was dissolved in water, dialyzed, andlyophilized as in 1. to give the product (320 mg) in 70% yield andcharacterized by ¹H NMR, sulfation SD=1.0-1.5.

9. Preparation of P-OSMeHA (DS-1) (GM-131101)

The TBA salt of MeHA (DS-1) (from MW 950 kDa HA) was prepared from 0.5mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1. Theresulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to which therequired excess (6 mol/equiv of available hydroxy groups in MeHA) ofpyridine-sulfur trioxide complex (0.8 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (510 mg)in 60% yield, which was shown by ¹H NMR to have a sulfation SD=1.0-1.5.

10. Preparation of P-OSFHA-1 (DS-2) (GM-231201)

The TBA salt of FHA-1 (FHA-1 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL of distilledwater and processing as above in 1. The resulting salt (FHA-1-TBA) wasdissolved in 25 mL of DMF to which the required excess (6 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.4g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated HA was dissolved in distilled water and dialyzedagainst 100 mM of NaCl solution for two days, changing the solution fourtimes a day, and lyophilized to give the product (280 mg) in 68% yieldand characterized by ¹H NMR, sulfation SD=1.0-1.5.

11. Preparation of P-OSFHA-2 (DS-2) (GM-331201)

The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of distilledwater and processing as above in 1. The resulting salt (FHA-2-TBA) wasdissolved in 25 mL of DMF to which the required excess (6 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.4g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated FHA-2 was dissolved in water, dialyzed, andlyophilized as in 1. to give the product (300 mg) in 69% yield andcharacterized by ¹H NMR, sulfation SD=1.0-1.5.

12. Preparation of P-OSMeHA (DS-2) (GM-131201)

The TBA salt of MeHA (DS-1) (from MW 950 kDa HA) was prepared from 0.5mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1. Theresulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to which therequired excess (6 mol/equiv of available hydroxy groups in MeHA) ofpyridine-sulfur trioxide complex (0.8 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (560 mg)in 64% yield, which was shown by ¹H NMR to have a sulfation SD=1.0-1.5.

13. Preparation of LMW-F-OSFHA-1 (DS-1) (GM-212101)

The TBA salt of LMW FHA-1 (FHA-1 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt(FHA-1-TBA) was dissolved in 25 mL of DMF to which the required excess(12 mol/equiv of available hydroxy group in HA) of pyridine-sulfurtrioxide complex (0.8 g) was added. After stiffing for 3 h at 40° C.,the reaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated HA was dissolved in distilled waterand dialyzed against 100 mM of NaCl solution for two days, changing thesolution four times a day, and lyophilized to give the product (300 mg)in 71% yield and characterized by ¹H NMR, sulfation SD=1.5-2.0.

14. Preparation of LMW-F-OSFHA-2 (DS-1) (GM-312101)

The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt (LMWFHA-2-TBA) was dissolved in 25 mL of DMF to which the required excess(12 mol/equiv of available hydroxy group in HA) of pyridine-sulfurtrioxide complex (0.8 g) was added. After stiffing for 3 h at 40° C.,the reaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated LMW FHA-2 was dissolved in water,dialyzed, and lyophilized as in 1. to give the product (260 mg) in 65%yield and characterized by ¹H NMR, sulfation SD=1.5-2.0.

15. Preparation of LMW-F-OSMeHA (DS-1) (GM-112101)

The TBA salt of LMW MeHA (DS-1) (from MW 53 kDa HA) was prepared from0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water as in 1.The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL of DMF to whichthe required excess (12 mol/equiv of available hydroxy groups in MeHA)of pyridine-sulfur trioxide complex (1.8 g) was added. After stirringfor 3 h at 40° C., the reaction was quenched by adding 100 mL of water,and crude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (480 mg)in 60% yield, which was shown by ¹H NMR to have a sulfation SD=1.5-2.0.

16. Preparation of LMW-F-OSFHA-1 (DS-2) (GM-212201)

The TBA salt of LMW FHA-1 (FHA-1 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt(FHA-1-TBA) was dissolved in 25 mL of DMF to which the required excess(12 mol/equiv of available hydroxy group in HA) of pyridine-sulfurtrioxide complex (0.8 g) was added. After stirring for 3 h at 40° C.,the reaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated HA was dissolved in distilled waterand dialyzed against 100 mM of NaCl solution for two days, changing thesolution four times a day, and lyophilized to give the product (300 mg)in 70% yield and characterized by ¹H NMR, sulfation SD=1.5-2.0.

17. Preparation of LMW-F-OSFHA-2 (DS-2) (GM-312201)

The TBA salt of LMW FHA-2 (FHA-2 from MW 53 kDa HA) was prepared byadding 0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL ofdistilled water and processing as above in 1. The resulting salt (LMWFHA-2-TBA) was dissolved in 25 mL of DMF to which the required excess(12 mol/equiv of available hydroxy group in HA) of pyridine-sulfurtrioxide complex (0.8 g) was added. After stirring for 3 h at 40° C.,the reaction was quenched by addition of 50 mL of water, and the crudematerial was precipitated by adding 75 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude partially O-sulfated LMW FHA-2 was dissolved in water,dialyzed, and lyophilized as in 1. to give the product (300 mg) in 68%yield and characterized by ¹H NMR, sulfation SD=1.5-2.0.

18. Preparation of LMW-F-OSMeHA (DS-2) (GM-112201)

The TBA salt of LMW MeHA (DS-1) (from MW 53 kDa HA) was prepared from0.5 mL of TBA and LMW MeHA (0.5 g) in 50 mL of distilled water as in 1.The resulting salt (LMW MeHA-TBA) was dissolved in 50 mL of DMF to whichthe required excess (12 mol/equiv of available hydroxy groups in MeHA)of pyridine-sulfur trioxide complex (1.6 g) was added. After stirringfor 3 h at 40° C., the reaction was quenched by adding 100 mL of water,and crude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (550 mg)in 63% yield, which was shown by ¹H NMR to have a sulfation SD=1.5.

19. Preparation of F-OSFHA-1 (DS-1) (GM-232101)

The TBA salt of FHA-1 (FHA-1 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL of distilledwater and processing as above in 1 The resulting salt (FHA-1-TBA) wasdissolved in 25 mL of DMF to which the required excess (12 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.8g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated HA was dissolved in distilled water and dialyzedagainst 100 mM of NaCl solution for two days, changing the solution fourtimes a day, and lyophilized to give the product (300 mg) in 68% yieldand characterized by ¹H NMR, sulfation SD=1.5.

20. Preparation of F-OSFHA-2 (DS-1) (GM-332101)

The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of distilledwater and processing as above in 1. The resulting salt (FHA-2-TBA) wasdissolved in 25 mL of DMF to which the required excess (12 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.8g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated FHA-2 was dissolved in water, dialyzed, andlyophilized as in 1. to give the product (310 mg) in 70% yield andcharacterized by ¹H NMR, sulfation SD=1.5-2.0.

21. Preparation of F-OSMeHA (DS-1) (GM-132101)

The TBA salt of MeHA (DS-1) (from MW 950 kDa HA) was prepared from 0.5mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1. Theresulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to which therequired excess (12 mol/equiv of available hydroxy groups in MeHA) ofpyridine-sulfur trioxide complex (1.6 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (500 mg)in 60% yield, which was shown by ¹H NMR to have a sulfation SD=1.5-2.0.

22. Preparation of F-OSFHA-1 (DS-2) (GM-232201)

The TBA salt of FHA-1 (FHA-1 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-1 (0.25 g) in 50 mL of distilledwater and processing as above in 1. The resulting salt (FHA-1-TBA) wasdissolved in 25 mL of DMF to which the required excess (12 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.8g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated HA was dissolved in distilled water and dialyzedagainst 100 mM of NaCl solution for two days, changing the solution fourtimes a day, and lyophilized to give the product (300 mg) in 69% yieldand characterized by ¹H NMR, sulfation SD=1.5-2.0.

23. Preparation of F-OSFHA-2 (DS-2) (GM-332201)

The TBA salt of FHA-2 (FHA-2 from MW 950 kDa HA) was prepared by adding0.5 mL of tributylamine to the FHA-2 (0.25 g) in 50 mL of distilledwater and processing as above in 1. The resulting salt (FHA-2-TBA) wasdissolved in 25 mL of DMF to which the required excess (12 mol/equiv ofavailable hydroxy group in HA) of pyridine-sulfur trioxide complex (0.8g) was added. After stirring for 3 h at 40° C., the reaction wasquenched by addition of 50 mL of water, and the crude material wasprecipitated by adding 75 mL of cold ethanol saturated with anhydroussodium acetate, and then collected by filtration. The resulting crudepartially O-sulfated FHA-2 was dissolved in water, dialyzed, andlyophilized as in 1. to give the product (300 mg) in 69% yield andcharacterized by ¹H NMR, sulfation SD=1.5-2.0.

24. Preparation of F-OSMeHA (DS-2) (GM-132201)

The TBA salt of MeHA (DS-1) (from MW 950 kDa HA) was prepared from 0.5mL of TBA and MeHA (0.5 g) in 50 mL of distilled water as in 1. Theresulting salt (MeHA-TBA) was dissolved in 50 mL of DMF to which therequired excess (12 mol/equiv of available hydroxy groups in MeHA) ofpyridine-sulfur trioxide complex (1.6 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude partially O-sulfated MeHA was dissolvedin water, dialyzed, and lyophilized as in 1 to give the product (550 mg)in 63% yield, which was shown by ¹H NMR to have a sulfation SD=1.5-2.0.

25. Preparation of P-OSBGHA (GM-431101)

The TBA salt of BGHA (from MW 950 kDa HA) was prepared from 0.5 mL ofTBA and BGHA (0.5 g) in 50 mL of distilled water as in 1. The resultingsalt (BGHA-TBA) was dissolved in 50 mL of DMF to which the requiredexcess (6 mol/equiv of available hydroxyl groups in BGHA) ofpyridine-sulfur trioxide complex (0.8 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude O-sulfated BGHA was dissolved in water,dialyzed, and lyophilized as in 1 to give the product (500 mg) in 60%yield, which was shown by ¹H NMR to have a sulfation SD<1.

26. Preparation of F-OSBGHA (GM-432101)

The TBA salt of BGHA (from MW 950 kDa HA) was prepared from 0.5 mL ofTBA and BGHA (0.5 g) in 50 mL of distilled water as in 1. The resultingsalt (BGHA-TBA) was dissolved in 50 mL of DMF to which the requiredexcess (12 mol/equiv of available hydroxyl groups in BGHA) ofpyridine-sulfur trioxide complex (1.6 g) was added. After stirring for 3h at 40° C., the reaction was quenched by adding 100 mL of water, andcrude material was precipitated by adding 150 mL of cold ethanolsaturated with anhydrous sodium acetate, and then collected byfiltration. The resulting crude O-sulfated BGHA was dissolved in water,dialyzed, and lyophilized as in 1 to give the product (520 mg) in 61%yield, which was shown by ¹H NMR to have a sulfation SD<1.

27. Preparation of P-OSBGHA (GM-411101)

The TBA salt of BGHA (from MW 53 kDa HA) was prepared from 0.5 mL of TBAand BGHA (0.5 g) in 50 mL of distilled water as in 1. The resulting salt(BGHA-TBA) was dissolved in 50 mL of DMF to which the required excess (6mol/equiv of available hydroxyl groups in BGHA) of pyridine-sulfurtrioxide complex (0.8 g) was added. After stirring for 3 h at 40° C.,the reaction was quenched by adding 100 mL of water, and crude materialwas precipitated by adding 150 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude O-sulfated BGHA was dissolved in water, dialyzed, andlyophilized as in 1 to give the product (500 mg) in 60% yield, which wasshown by ¹H NMR to have a sulfation SD<1.

28. Preparation of P-OSBGHA (GM-412101)

The TBA salt of BGHA (from MW 53 kDa HA) was prepared from 0.5 mL of TBAand BGHA (0.5 g) in 50 mL of distilled water as in 1. The resulting salt(BGHA-TBA) was dissolved in 50 mL of DMF to which the required excess(12 mol/equiv of available hydroxyl groups in BGHA) of pyridine-sulfurtrioxide complex (1.6 g) was added. After stirring for 3 h at 40° C.,the reaction was quenched by adding 100 mL of water, and crude materialwas precipitated by adding 150 mL of cold ethanol saturated withanhydrous sodium acetate, and then collected by filtration. Theresulting crude O-sulfated BGHA was dissolved in water, dialyzed, andlyophilized as in 1 to give the product (500 mg) in 60% yield, which wasshown by ¹H NMR to have a sulfation SD<1.

III. Preparation of Fluorescent SAGEs and Palmitoylated SAGE

a. Preparation of LMW-F-OSFHA-1 (DS-2) Fluorescent Conjugate

LMW-F-OSFHA-1 (50 mg) and NHS (40 mg) were dissolved in 10 mL water, andthen Alaxa fluo @488 (1 mg) in 4 mL DMF was added. And then pH wasadjusted to 4.75. after that, 100 mg EDCI was added in solid form. ThepH was kept at 4.75 by adding NaOH solution. The solution was stirredfor ovenight at room temperature with aluminum foil coverage. Then theproduct was purified by dialyzing against distilled water (cut-off MW3500) and following methanol/water solution (50/50, v/v). furtherpurified by gel filtration column (Sephadex G-25). Further purified byPD-10 column and lyophized to dry to obtain 40 mg.

b. Preparation of LMW-P-OSMeHA (DS-1) fluorescent conjugate

LMW-P-OSMeHA (50 mg) and NHS (40 mg) were dissolved in 10 mL water, andthen Alaxa fluo @488 (1 mg) in 4 mL DMF was added. And then pH wasadjusted to 4.75. after that, 100 mg EDCI was added in solid form. ThepH was kept at 4.75 by adding NaOH solution. The solution was stirredfor ovenight at room temperature with aluminum foil coverage. Then theproduct was purified by dialyzing against distilled water (cut-off MW3500) and following methanol/water solution (50/50, v/v). furtherpurified by gel filtration column (Sephadex G-25). Further purified byPD-10 column and lyophized to dry to obtain 43 mg.

c. Preparation of Palmitoylated SAGE

LMW-P-OSMeHA (50 mg) was dissolved in 50 mL of DMF, triethylamine (0.3mL) was added to the DMF solution while stirred. After 5 min, thepalmitoyl chloride (0.5 mL) was added. The resulting mixture was keptstiffing for overnight. The solution was evaporated, and the residue wasdissolved in distilled water, dialyzed for one day (change water fourtimes), and lyophilized to dry to obtained 35 mg.

IV. SAGEs are Potent Inhibitors of P-selectin, Human Leukocyte Elastaseand the Interaction of RAGE with all of its Ligands

Materials. Polyclonal goat anti-human RAGE, recombinant human highmobility box protein-1 (HMGB-1), recombinant human P-selectin/Fcchimera, recombinant human RAGE/Fc chimera, human azurocidin andpolyclonal goat anti-human azurocidin were purchased from R&D Systems(Minneapolis, Minn.). Human S100b calgranulin was from Calbiochem (SanDiego, Calif.). The advanced glycation end-product carboxymethyllysine-bovine serum albumin (CML-BSA) was obtained from MBLInternational (Woburn, Mass.). U937 human monocyte cells were obtainedfrom American Type Culture Collection (Manassas, Va.). Protein A, horseradish peroxidase-conjugated rabbit anti-goat IgG, carbonate-bicarbonatebuffer and bovine serum albumin blocker (10×) were obtained fromPiercenet (Rockford, Ill.). Calcein AM, Dulbecco's modified Eagle'smedium (DMEM), ethylenediamine tetraacetic acid (EDTA), fetal bovineserum (FBS), HEPES, non-essential amino acids,penicillin/streptomycin/L-glutamine solution, RPMI-1640 withoutL-glutamine and sodium bicarbonate were obtained from Invitrogen(Carlsbad, Calif.). High-bind 96-well microplates were obtained fromCorning Life Sciences (Corning, N.Y.). All other chemicals not specifiedwere purchased from Sigma-Aldrich (St. Louis, Mo.).

Cell Culture. U937 monocytes were grown in suspension culture at 37° C.in humidified 5% CO₂-95% air in RPMI-1640 supplemented with 10% heatinactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM MEMnon-essential amino acids, 100 units/ml penicillin and 100 mg/mlstreptomycin. Experiments were performed on cells from passages 1-5.

Cell Binding Assays. The effect of SAGEs on binding of U937 monocytes toP-selectin or RAGE was studied in high-bind micro plates coated with 8μg/ml protein A (50 μg/well) in 0.2 M carbonate-bicarbonate buffer (pH9.4). Plates were washed with phosphate buffered saline containing 1%BSA (PBS-BSA), and P-selectin-Fc or RAGE-Fc chimera (50 μL containing 1μg) was added to each well and incubated for 2 h at room temperature orovernight at 4° C., respectively. Following incubation, wells werewashed twice with PBS-BSA. Fifty (50) μL of SAGEs (0 to 1,000 μg/ml)serially diluted in 20 mM HEPES buffer (containing 125 mM NaCl, 2 mMcalcium and 2 mM magnesium) were added to each well and incubated atroom temperature for 15 min. As a negative control, 50 μL of 10 mM EDTAwas added to select wells to prevent cell binding through sequestrationof calcium. At the end of the incubation period, 50 μL of U937 cells(10⁵ cells/well, calcein-labeled according to manufacturer'sinstructions) were added to each well and plates were incubated anadditional 30 min at room temperature. The wells were then washed thricewith PBS, and bound cells were lysed by addition of 100 μL ofTris-TritonX-100 containing buffer. Fluorescence was measured on amicroplate reader using excitation of 494 nm and emission of 517 nm.

Solid Phase Binding Assays. Solid phase binding assays were used tostudy the ability of SAGEs to inhibit RAGE binding to its ligands. Forstudies of the effect of SAGEs on RAGE binding to its ligands, polyvinyl96-well plates were coated with 5 μg/well of specific ligand (CML-BSA,HMGB-1 or S100b calgranulin). Plates were incubated overnight at 4° C.and washed thrice with PBS-0.05% Tween-20 (PBST). Separately, RAGE-Fcchimera (100 μL containing 0.5 μg/ml in PBST-0.1% BSA) was incubatedwith an equal volume of serially diluted SAGEs (0.001 to 1,000 μg/ml inPBST-BSA) overnight at 4° C. The following day, 50 μL of RAGE-SAGE mixwas transferred to each respective ligand-coated well and incubated at37° C. for 2 h. Wells were then washed four times with PBST. To detectbound RAGE, 50 μL of anti-RAGE antibody (0.5 μg/ml) was added to eachwell, the mixture was incubated for 1 h at room temperature, and wellswere washed again four times with PBST. Horse-radish peroxidaseconjugated secondary antibody (1:10,000 antibody dilution in PBST; 50 μLper well) was added, wells were incubated for 1 h at room temperature,and then washed once with PBST. A colorimetric reaction was initiated byaddition of 50 μL of tetramethyl benzidine chromogen (TMB singlesolution chromogen) and terminated after 15 min by addition of 50 μL of1 N HCl. Absorbance at 450 nm was read using an automated microplatereader.

Enzymatic Assays. To characterize SAGE inhibition of the cationic PMNprotease HLE an activity assay (Fryer A, Huang Y-C, Rao G, Jacoby D,Mancilla E, Whorton R, Piantadosi C A, Kennedy T, Hoidal J. SelectiveO-desulfation produces nonanticoagulant heparin that retainspharmacologic activity in the lung. J Pharmacol Exp Ther 282:208-219,1997) was employed, which measured the ability of purified HLE to cleavea chromogenic substrate. HLE (100 nM) was incubated with SAGE (1-100 nM)in 0.5 M HEPES buffer for 15 min. Following incubation, the elastasesubstrate Suc-Ala-Ala-Val-p-nitroanaline (p-NA) was added to thereaction mixture to a final concentration of 0.3 mM. The hydrolysis ofp-NA released was followed for 15 min by measuring the absorbance at 405nm. In order to characterize the ability of SAGEs to activate Factor XIIor complement, activity assays were employed that are similar to thoserecently used to screen for toxicity of adulterated commercial heparin(Kishimoto T K, Viswanathan K, Ganguly T, Elankumaran S, Smith S, PelzerK, Lansing J C, Sriranganathan N, Zhao G, Galcheva-Gargova Z, Al-HakimA, Bailey G S, Fraser B, Roy S, Rogers-Cotrone T, Buhse L, Whary M, FoxJ, Nasr M, Dal Pan G J, Shriver Z, Langer R S, Venkataranam G, Austen KF, Woodcock J, Sasisekharan R. Contaminated heparin associated withadverse clinical events and activation of the contact system. N Engl JMed 358:2457-2467, 2008; Guerrini M, Beccati D, Shriver Z, Naggi A,Viswanathan K, Bisio A, Capita I, Lansing J C, Guglieri S, Fraser B,Al-Hakim A, Gunay N S, Zhang Z, Robinson L, Buhse L, Nasr M, Woodcock J,Langer R, Venkataraman G, Linhardt R J, Casu B, Torri G, Sasisekharan R.Oversulfated chondroitin sulfate is a contaminant in heparin associatedwith adverse clinical events. Nat Biotech 26:669-675, 2008). Pooledhuman plasma (5 μl) was incubated with 100 μl of SAHA (0.1 to 1000μg/ml) in 0.05 M HEPES containing Triton X-100 for 5 min at 5° C.Amidolytic activity specific for Hageman factor was determined by adding0.5 mM D-cyclohydrotyrosyl-Glyc-L-Arg-p-NA and following the change inabsorbance at 405 nm (Silverberg M, Dunn J T, Garen L, Kaplan A P.Autoactivation of human Hageman factor. Demonstration using a syntheticsubstrate. J Biol Chem 255:7281-7286, 1980). Amidolytic activityspecific for active kallikrein was determined by addingD-Pro-Phe-Arg-p-NA and following change in absorbance at 450 nm.

Results: The results of the assays are shown in Table 2. SAGEs arepotent inhibitors of P-selectin. The competitor-mediated displacement ofU937 human monocytes, which firmly adhere to P-selectin throughP-selectin glycoprotein ligand-1 (PSGL-1), was studied usingfluorescently-labeled cells. FIG. 3 shows that a SAGE inhibits U937binding to P-selectin with a 50% inhibitory concentration (IC₅₀) of 0.5μg/ml.

TABLE 2 50% Inhibitory Concentrations (IC₅₀) in μg/ml RAGE/CML-Leukocyte Hageman SAGE p-Selectin/PSGL RAGE/Mac-1 BSA RAGE/S100BRAGE/HMGB1 Elastase Factor GM-211101 GM-311101 GM-111101 0.017 0.0330.082 0.12 0.58 0.4 GM-211201 NR NR NR NR 0.54 NR GM-311201 NR NR NR NR0.52 NR GM-111201 0.14 0.042 2.27 1.56 0.22 na GM-231101 NR 14.6GM-331101 NR 4.557 NR GM-131101 GM-231201 8.82 GM-331201 2.28 GM-1312010.5 0.3 0.044 0.06 1.66  0.42 0.4 GM-212101 GM-312101 0.56 GM-1121010.002 0.042 GM-212201 GM-312201 0.036 0.009 0.059 0.075 0.47 0.4GM-112201 GM-232101 0.041 0.02 0.408 GM-332101 0.015 0.021 0.371GM-132101 GM-232201 GM-332201 0.22 GM-132201 0.22 0.004 0.1 0.04 0.240.4 GM-431101 89.5 GM-432101 0.85 GM-411101 GM-412101

Second, as highly sulfated polyanions, alkylated and sulfated SAGEs arepotent inhibitors of polymorphonuclear leukocyte proteases. FIG. 4 showsthat alkylated and/or fluoroalkyated and sulfated SAGEs inhibit humanleukocyte elastase (HLE) with impressively potent IC₅₀ values.Specifically, the non-alkylated, fully O-sulfated HA (F-OSHA) shows an0.66 nM IC₅₀ for HLE. For the modified and sulfated HA derivatives, IC₅₀values were 1.89 nM for partially O-sulfated carboxymethylated HA(P-OSCHMHA); 1.97 nM for partially O-sulfated HA (P-OSHA); and 3.46 nMfor partially O-sulfated methylated HA (P-OSMEHA; GM-131101).

Third, SAGEs are extremely potent inhibitors of RAGE. SAGEs inhibitedthe interaction of RAGE and amphoterin (HMGB-1) with an IC₅₀ of 1.1μg/ml (FIG. 5), the interaction of RAGE and S100 calgranulins with anIC₅₀ of 60 ng/ml (FIG. 6), and the binding of RAGE to the AGE productcarboxymethyl-lysine BSA with an IC₅₀ of 44 ng/ml (FIG. 7). These valuesare 5 to 10-fold more potent than corresponding levels of RAGE-ligandinhibition we have measured with heparin and 2-O, 3-O desulfatedheparin.

Fourth, SAGEs are potent inhibitors of the proliferation of humankeratinocytes. In these assays human neonatal epidermal keratinocyteswere cultured in the presence and absence of SAGEs or other inhibitors,and proliferation was measured by adding a dye that is reduced in directproportion to the number of viable cells present. In the figuresdisplayed, absorbance values were normalized to controls, which wereassigned a relative absorbance of 1.0. FIG. 8 shows that the highmolecular weight partially O-sulfated HA (P-OSHA) and fully O-sulfatedHA (F-OSHA) were more effective than the heparin derivative 2-O, 3-Odesulfated heparin (ODSH) at preventing keratinocyte proliferation whenadded in concentrations of 100 μg/ml. Higher sulfation appearsadvantageous, since partially sulfated carboxymethylated HA (P-OSCMHA)and methylated HA (P-OSMEHA; GM-131101) were less active at inhibitingkeratinocyte proliferation. FIG. 9 shows that overall lower molecularweight (LMW) derivatives were more effective than higher molecularweight derivatives at reducing keratinocyte proliferation in this assay.In particular, the LMW partially O-sulfated MeHA (LMW-P-OMEHA;GM-111201), partially O-sulfated CMHA (LMW-P-OSCMHA) and partiallyO-sulfated 53 kDa HA itself (LMW-P-OSHA) all reduced proliferationrelative to controls at concentrations as low as 1 μg/ml.

V. In Vivo Studies for the Treatment of Rosacea and Inflammation

Materials and Methods

Chemicals. GM-111101, GM-131101, GM-312101 and GM-212101 were evaluated.Medium was bought from American Type Culture Collection (ATCC). EpiLifemedium was purchased from Invitrogen (Madison, Wis.).

Cells. The Human Dermal Fibroblasts (nHDF) were purchased from ATCC.Human neonatal epidermal keratinocytes (HEKn) were obtained fromInvitrogen (Madison, Wis.).

Cytotoxicity. 4,000 nHDF cells were seeded 100 μl medium in each well of96-well flat-bottomed microplates, and incubated at 37° C. in 5% CO₂ for12 hours. All the medium was changed with complete medium containing avariety of sulfated hyaluronan (HA) derivatives at final concentrationsof 10, 100, 1000, 10000, 100000, 1000000 ng/ml to each column. At 48hours, 20 μl MTS (Promega, Madison, Wis.) was pipetted into each well,and cells were further incubated for 2 hours. The absorbance of thesamples was measured at 490 nm using a 96-well plate reader.

Skin Irritation Test in Mice. GM-111101 and GM-212101 were tested invivo to determine the dermal irritation potential to the skin of themice. The two test agents were prepared with two differentconcentrations individually, 0.1 mg/ml and 1 mg/ml. 10% formic acid andPBS were used as positive and negative control respectively (n=6).Balb/c mice, which had not been used in previous experiments and wereobserved to be free from any skin irritation, trauma, or adverseclinical signs prior to initiation of the studies, were randomized andgrouped for designed test conditions. The back of the animals wasclipped free of fur with an electric clipper at least 4 hours beforeapplication of the sample. Just prior to test substance application,each mouse received four parallel epidermal abrasions with a sterileneedle at the bottom area of the test site while the upper area of thetest site remained intact. Under anesthesia, two 0.5-ml samples of thetest solution were applied to the entire test site by introduction undera double gauze layer to an area of skin approximately 2.5 cm square. Thepatches were backed with plastic, covered with a non reactive tape andthe entire test site wrapped with a bandage. Animals were returned totheir cages. After a 24 hr exposure to the agent, the bandage and soakedtest gauze were removed. The test sites were wiped with tap water toremove any remaining test compound. At 24 and 72 hours after compoundapplication, the test sites were examined for dermal reactions inaccordance with the FHSA-recommended Draize scoring criteria. ThePrimary Irritation Index (P.I.I.) of the test article will be calculatedfollowing test completion. A material producing a P.I.I. score ofgreater than or equal to 5.00 would be considered positive; the materialwould be considered a primary irritant to the skin.

Animals. Balb/c mice were purchased commercially from a vendor approvedby the Univ. of Utah veterinary medicine department and vivarium. Afterthey are quarantined for the prescribed period following receipt, theyare ready for use.

LL37 Peptide and SAGE Injection Roceasea Models. Chronic disease of theskin leaves an indelible mark on the patient's life, especially, as inthe case of rosacea, when it presents on the face. As a species, wereact positively or adversely to one another's appearance, instinctivelyrecoiling from those who appear abnormal and unlike ourselves. Whileskin diseases are not often life-threatening, they are life-altering inways that normal individuals do not fathom. Rosacea is one of thoselife-altering illnesses. Rosacea is a common disfiguring facial skindisease affecting 3% of the U.S. population, or about 14 millionAmericans, with onset usually between the ages of 30 and 50 years. Itstrikes primarily in Caucasians of Celtic descent, and appears to bemore problematic and common in women than men. Over a third of patientshave a family history, strongly suggesting an inherited illness. Thecondition is particularly stigmatizing because of the commonmisconception that the facial redness and the knobby nose of rosacea arethe consequence of excessive alcohol consumption. Rosacea presents asseveral clinical phenotypes. The most common presentation ischaracterized by transient or persistent central flushing and erythemaof the face, with dilated capillaries on the cheeks and nose. Thisphenotype ranges from a “ruddy complexion” to persistent, readilyvisible dilated vessels. This subtype is without an effective topicaltreatment. In a second common presentation, papules and pustules occuron the central convexities of the face, frequently superimposed upon abackground of erythema. On biopsy, the skin is richly infiltrated withPMNs. Papulopustular rosacea is treated empirically with topical orsystemic antiobitics, but is not clearly related to documented cutaneousinfection. Macrolides, either topically or systemically, been most oftenused as therapy, and may produce improvement not because of theiranti-bacterial activity so much as their ability to retard PMNchemotaxis into areas of inflammation. In a third and fortunately morerare phenotype, rosacea produces rhinophyma from hyperplasia of both thesebaceous glands and connective tissue of the nose, creating theclassical “W. C. Fields” nose, or “whiskey nose”. Treatment of thiscondition requires surgical excision of the tissue, or laser therapy, toremove hypertrophied tissue. In a fourth phenotype, rosacea can producedry, itchy eyes with irritation of the lids (blepharitis),photosensitivity, blurred vision and conjunctivitis. Prolonged diseasecan result in keratitis and even corneal scarring. This phenotype, whicharises from inflammation of the lids and the meibomian glands on thelower lid surface, is common and overlaps clinically with the “dry-eye”syndrome seen frequently by ophthalmologists. The occurrence of rosaceaexclusively on sun-exposed facial areas in those with fair skin andlight eyes points to a pathogenic role for solar radiation in causingthe condition.

The pathogenesis of rosacea has recently been elucidated (Yamasaki K, DiNardo A, Bardan A, Murakami M, Ohtake T, Coda A, Dorschner R A, BonnartC, Descargues P, Hovnanian A, Morhenn V B, Gallop R L. Increased serineprotease activity and cathelicidin promotes skin inflammation inrosacea. Nat Med 13:975-980, 2007; Bevins C L, Liu F-T. Rosacea: skininnate immunity gone awry? Nat Med 13:904-906, 2007). In elegant workYamasaki et al. demonstrated that the skin involved with rosaceacontains high levels of cathelicidins and their processing proteaseSCTE. Cathelicidins, a major family of antimicrobial peptides inmammals, are expressed in leukocytes and epithelial cells of manyorgans, where they mediate innate immune responses to bacteria (Nizet V,Ohtake T, Lauth X, Trowbridge Jk, Rudisill J, Dorschner R A,Prestonjamasp V, Piraino J, Huttner K, Gallop R L. Innate antimicrobialpeptide protects the skin from invasive bacterial infection. Nature414:454-457, 2001). Cathelicidins also signal vascular growth, PMNmigration and wound healing (Bevins, ibid). Human cathelicidin issecreted by keritinocytes as an 18-kDa hCAP18 pro-peptide and cleaved bySCTE to the C-terminal active anti-microbial peptide LL-37, designatedafter its 37 amino acid length and two N-terminal leucines. Skininvolved with rosacea demonstrates inappropriately high levels of bothcathelicidins and SCTE without clearly inciting microbial invasion as astimulus. When placed in the culture media covering primary humankeritinocytes LL-37 greatly augments production of chemotactic cytokinessuch as IL-8. When injected intradermally into mice in levels similar tothose found in rosacea-involved skin, LL-37 produces erythema andstimulates prominent dermal infiltration by PMNs. Intradermal injectionof SCTE also produces skin erythema and PMN infiltration of the dermisin wild type, but not in cathelicidin^(−/−) mice. Thus, there is agrowing body of evidence which supports the concept that rosacea ismediated by local over-expression of pro-inflammatory, cationic skinpeptides which produce the inflammation, excessive angiogenesis andsebaceous hyperplasia characteristic of the disease. Because nearlyevery patient with rosacea can tell which of his parents has a patternof reactive facial flushing and blushing, it is apparent to cliniciansthat rosacea is genetically driven.

To produce a model of rosacea, LL-37 was injected intradermally every 12h for 48 h. This model produced erythema of the skin and prominentintradermal infiltration of polymorphonuclear leukocytes (PMNs), asreported by Yamasaki et al. Balb/c mice were shaved prior to study toexpose an area of skin on the back. Twenty-four hours later, we injected40 μuL of vehicle (phosphate buffered saline, PBS), cationic peptide (at320 μM concentration in PBS), SAGE (320 to 1,280 μM in PBS), or cationicpeptide+SAGE mix (peptide+1× to 4× molar concentrations of SAGE)intradermally into the shaved skin using a 31 gauge needle in a mannerdesigned to raise an intact epidermal bleb, thereby identifying thatadministration was at the level of the lower epidermis or dermis. SAGEselected for injection were chosen from over twelve newly synthesizedSAGEs, which were tested extremely active in biochemical assays asinhibitors of human leukocyte elastase (as another cationic protein) andas antagonists for activation of the receptor for advanced glycationend-products (RAGE) by four common ligands.

SAGEs and peptides were mixed together in PBS prior to injection andallowed to incubate 15 min at room temperature before being injected.Injections were repeated every 12 h thereafter. Forty-eight hours afterthe initial injection (four injections in total), animals were lightlyanesthetized with 25 mg/kg pentobarbital intraperitoneally. When themouse was asleep, the area of injected skin was photographed to visuallyrecord the severity of erythema and edema. The intensity of erythema wasassessed as a redness score (from 1 to 5), and the area of erythema wasmeasured with calipers. The area of injected skin was then excisionallybiopsied using a 6 mm hole punch for hematoxylin-eosin staining toexamine the histopathologic changes and to assess PMN infiltrationthrough measurement of myeloperoxidase (MPO) activity. Onerepresentative image of skin surface and histology from each skin wasviewed under high power viewing under a microscope.

SAGE Topical Treatment Rosacea Model. Balb/c mice were shaved in timefrom LL37 exposure on area of skin on the back. We then began topicalapplication of a hyaluronan-cased emollient containing 5% SAGEs (activeemollient) or hyaluronic acid based emollient alone to this area of skinevery 12 hours. Twenty-four hours later, we injected 40 μL of vehicle(PBS) or cationic peptide (at 320 μM concentration) subcutaneously intothe shaved skin in the manner described previously. Injections andtopical emollient applications were repeated every 12 h thereafter.Forty-eight hours after the initial injection (four injections intotal), animals were lightly anesthetized as described previously. Thearea of injected skin was photographed to visually record the severityof erythema and edema. The intensity of erythema was assessed as aredness score (from 1 to 5), and the area of erythema was measured withcalipers. The area of injected skin was then excisionally biopsied usinga 6 mm hole punch for H&E staining to examine the histopathologicchanges and to assess PMN infiltration through measurement ofmyeloperoxidase (MPO) activity.

SAGE Dermis Penetration. Balb/c mice were shaved prior to study toexpose an area of skin on the back. Topical application of SAGE wascarried out to the skin every 12 h. Forty-eight hours later animals wereeuthanized and the skin was biopsied. Sections of skin were then studiedby fluorescence microscopy to determine the depth to which SAGEspenetrate into the skin.

Croton Oil Inflammation Model. As another model of PMN-mediated skininflammation, croton oil was employed. Croton oil contains phorbolesters, which activate protein kinase C in skin cells. As a result, skincell produce abundant chemokines and chemotaxins which signal the influxof PMNs from the circulation. Activated PMNs produce erythema and edemaof skin tissues. Croton oil induced inflammation is a commonly employeemodel of PMN-mediated skin inflammation in the screening ofanti-inflammatory compounds for dermatologic use.

To produce this model, croton oil (Sigma-Aldrich, St. Louis, Mo.) wasmixed as a 0.8% solution in acetone. Using a pipette, 10 μl were paintedonto each side of one ear of the mouse, with the other ear remaining asa control. At 4, 8 and 24 hours later, ear thickness was measured nearthe top of the ear distal to the cartilaginous ridges. Change in earthickness from control was then taken as an index of edema. Theintensity of erythema was assessed as a redness score (from 1 to 5), andthe area of erythema was measured with calipers. Following the 24 hourmeasurements, mice were euthanized and ear punch biopsies (6 mm holepunch) were taken immediately, weighted, frozen and stored at −80° C.for H&E staining to examine the histopathologic changes and to assessPMN infiltration through measurement of myeloperoxidase (MPO) activity.A single investigator performed all ear measurements and biopsies inorder to standardize the procedure and reduce error. The remainder ofears were removed, embedded and frozen for immunohistochemistry.

Myeloperoxidase (MPO) Assay. For each mouse, tissue biopsies (6 mmdiameter hole punch) were taken immediately, weighted, frozen and storedat −80° C. Tissue MPO activity was measured using a method by Suzukiet.al. (Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assaymethod for myeloperoxidase in human polymorphonuclear leukocytes. AnalBiochem 132:345-352, 1983) as modified by Young et. al. (Young J M,Spires D A, Bedord C J, Wagner B, Ballaron S J, De oung L M. The mouseear inflammatory response to typical arachidonic acid. J Invest Dermatol82:367-371, 1984). Each mouse tissue biopsy was placed in 0.75 mL of 80mM phosphate-buffered saline (PBS) pH 5.4 containing 0.5%hexadecyltrimethyl-ammonium bromide (HTAB). Each sample was homogenizedfor 45 s at 4° C. with a small laboratory Tissue Tearor HomogenizerModel 985-370 (Biospec Products, Bartlesville, Okla.). The homogenatewas transferred quantitatively to a microcentrifuge tube with anadditional 0.75 mL HTAB in PBS. The 1.5 mL sample was centrifuged at12,000×g for 15 min, maintained at 4° C. Triplicate 30 uL samples of theresulting supernatant were added to 96-well microtier plate wells. Forthe MPO assay, 200 uL of a mixture containing 100 uL of 80 mM PBS (pH5.4), 85 uL of 0.22 M PBS (pH 5.4), and 15 uL of 0.017% hydrogenperoxide were added to each well. 20 uL of 18.4 mM tetramethylbenzidineHCl in 8% aqueous dimethylformamide was added to start the reaction.Microtiter plates were incubated at 37° C. for 3 min, and then placed onice. The reaction was stopped with the addition of 30 uL of 1.46 Msodium acetate. MPO enzyme activity was assessed at an absorbancewavelength of 630 nm. MPO activity was expressed as optical density(OD)/biopsy.

Statistical Analyses. All experiments were performed in triplicate forin vitro tests. Significance differences between samples were calculatedby comparison of means using the Aspin-Welch test. Significance wasdeclared at p<0.05.

Results

LL37 Peptide and SAGE Injection Roceasea Models. To determine if directneutralization of cationic cathelicidins prevented their inflammatoryactivity in the skin, LL37 only, SAGE (GM-111101) only, vehicle (PBS)only, or mixture of LL37 and SAGE were subcutaneously injected into theshaved back area of mice every 12 h thereafter. After 48 hours, micewere sacrificed and gross pictures in different treatment groups weretaken (FIGS. 10 a and 10 b). Histological studies using hematoxylin andeosin staining showed increased number of leukocytic infiltration andmarked dermal edema, whereas SAGE administration immediately afterchallenge resulted in the inhibition of skin swelling response (FIGS. 10c and 10 d).

Individual results of dermal scoring were expressed by erythema area(FIG. 10 f) and erythema redness score (FIG. 10 g). After 48 hours, theSAGE treated group demonstrated a dramatically decreased area oferythema and a significant reduction of redness score. Myeloperoxidaseactivity was measured in the tissue punch biopsies taken 48 hr afterinjection as an index of PMN infiltration. SAGE coadministration withLL-37 peptide significantly reduced MPO activity by 50% (FIG. 10 e).Therefore, co-injection of SAGE with LL-37 peptide substantially inducedthe inflammatory activity of the LL-37 cathelicidin peptide. Thisindicates that SAGEs inhibit LL-37 mediated inflammation and would beuseful treatments for rosacea.

SAGE Topical Rosacea Treatment Model. Topical treatment of SAGE(GM-111101) is used to test if treatment remote in time from LL-37exposure can also prevent peptide-induced skin inflammation. Therefore,after the LL-37 injection into the mouse back skin area, SAGE wasapplied right after. The gross pictures showed strong edema and erythemaat 48 hours after the LL-37 application (FIGS. 11 a, 11 h, and 11 i),while topical treatment with SAGE significantly decreased the rednessand its affected area both for immediately treatment (FIG. 11 b) and 12h delayed treatment (FIG. 11 c). The H&E staining indicated much moreleukocytic infiltration and dermal edema than the two SAGE treatmentgroups, which was in agreement with the results of SAGE as inhibitor ofskin swelling response and MPO activities (FIG. 11 g). These resultsindicate that SAGEs can be applied topically in a conventional andpharmaceutically acceptable emollient to treat the cathelicidin-mediatedinflammation of rosacea.

SAGE Dermis Penetration. To determine the level of which SAGE penetratesinto the dermis. SAGE compound fluorescent-GM-212101 andfluorescent-GM-111101 were used as test article, and 0.1 mg/ml, 1 mg/mland 10 mg/ml fluorescent compound were applied on the abraded anduntouched skin area of Balb/c mice. After 24 hours, mice were sacrificedand the whole tested skin area was excised and photographed under bothnatural light and long wavelength UV light condition. (FIG. 12) Layersof fluorescence were observed under both natural and UV light conditionfor both inner and outer treatment area skin. Significant penetration ofSAGEs was distributed even on a micrometer-length scale.

Cytotoxicity and Skin Irritation In Vivo Tests. The cytotoxicity of SAGEderivatives (SAGEs) GM-131101, GM-312101 and GM-212101 was evaluated innHDF cells and the results are demonstrated in FIG. 13 a. All compoundswere also found to be non-toxic to the nHEK cells up to 10 mg/mlconcentration (FIG. 13 b). For the in vivo skin irritation tests, grosspictures of mice in different treatment groups were represented in FIG.13 c-13 j. The Primary Irritation Index of the test substances wascalculated to be 0.00 for both GM-111101 and GM-212101; No irritationwere observed on the skin of the mice (FIG. 14). Both the GM-111101 andGM-212101 have not been found cytotoxic. The concentration threshold ofall SAGEs could be determined from this test. Under the conditions ofthis test, the test agents would not be considered a primary skinirritant; as defined in the guidelines of the FHSA Regulations, 16 CFR1500, a substance with an empirical score of less than 5.00 is not aprimary irritant to the skin. These results indicate that SAGEs arenon-irritating themselves for skin and can be employed as safetreatments for inflammatory skin disorders.

Croton Oil Inflammation Model. The application of croton oil to mouseskin was used as a convenient and highly reproducible model ofPMN-mediated skin inflammation. This model was used to test theanti-inflammatory activity of SAGEs. Gross pictures of mice in differenttreatment groups were represented in FIGS. 15 a and 15 b, as well as theear thickness measured for both the treated and untreated ears, andcompared in all the five groups. Individual results of dermal scoringwere expressed by erythema for abraded area and intact area. The resultsshowed significant reduction of redness and thickness in the SAGE(GM-111101) treatment groups compared with non-treatment groups (FIGS.15 g and 15 h). Histopathological exams revealed that in croton oilpainted ears, there was an increased number of leukocytic infiltrationand marked dermal edema, whereas SAGE administration immediately afterchallenge resulted in the inhibition of ear swelling response, which wascomparable to that of vehicle-treated mice. These histological findingsfurther confirmed those of the measurement data. MPO activity was alsomeasured in the ear punch biopsies taken after croton oil application.SAGE treatment every 4 hr starting immediately after croton oilapplication significantly reduced MPO activity (FIG. 15 f). Theseresults indicate that SAGEs can be employed as topical treatments forinflammatory skin disorders other than rosacea.

Hyaluronic Acid (HA) Topical Treatment in Rosacea Model. The previoustopical treatment of LL-37 rosacea model was used to compare SAGE(GM-111101) vs. HA. The results clearly showed that HA in the topicaladministration did not alleviate inflammation (FIGS. 16 b, 16 e, and 7f) and MPO activities (FIG. 16 d). Conversely, SAGE possessed highlyenhanced anti-inflammatory properties (FIG. 16 c-f) and could beconsidered as an inhibitor of inflammatory and rosacea. These dataindicate that the pharmacologic activity of SAGEs is not inherent inhyaluronic acid, but require the novel chemical modifications ofhyaluronan.

In Vivo Acute Intraveneous Toxicity Study in Rats. The objective of thestudy was to evaluate the acute intravenous toxicity of SAGEs GM-111101and GM-212101 when administered as a single dose to rats and also toevaluate the toxicity of GM-111101 when administered once daily for aperiod of seven days at a single dose level.

One dose group consisting of three male and three female Sprague-Dawleyrats was exposed to a single dose of 3 mg/kg GM-111101, then a singledose of 10 mg/kg GM-111101 one week following and finally once dailydoses of 10 mg/kg GM-111101 for a total of seven days initiating oneweek following the last single dose. Two groups of three male and threefemale Sprague-Dawley rats were exposed to GM-111101 at doses of 30 or100 mg/kg. In addition, two groups of three male and three femaleSprague-Dawley rats were exposed to GM-212101 at doses of 30 or 100mg/kg. Two groups of three male and three female Sprague-Dawley ratswere exposed to 0.9% Sodium Chloride for Injection and were used asnegative control groups. All doses were administered at a dose volume of1 mL/kg by intravenous injection via the caudal tail vein. Dosecalculations were determined based upon the most recently documentedbody weight.

All animals exposed acutely (single dose) were observed immediatelyfollowing injection, and again at 2 hours and 4 hours following doseadministration on Day 0 for apparent signs of clinical toxicity. Inaddition, all surviving animals were observed once daily on days 1-14for apparent signs of clinical toxicity. All animals exposed once dailyfor seven days were observed once daily from days 1-14 for apparentsigns of clinical toxicity. Body weight was recorded on day 0 (Acutedose) or day 1 (Repeat dose) prior to dose administration, on day 6 or7, and on day 14, prior to termination. Gross necropsy evaluations wereperformed on each of the surviving animals on day 14 of the study.Animals that died on study underwent a gross necropsy examinationimmediately following observation of mortality.

Clinical signs of moderate abnormal gait, moderate ataxia, andreddish-orange discolored urine were observed in one female animal fromthe 30 mg/kg GM-212101 dose group within the first 2 hours followingdose administration. These observations, with the exception of mildataxia, were no longer present as of the 4 hour observation period andremained that way throughout the remainder of the study. The observationof ataxia was no longer present on the day following doseadministration. In addition, one male rat from the 100 mg/kg GM-212101dose group was found dead within the first four minutes following doseadministration. All animals gained body weight throughout the studyperiod. There were no visible lesions observed at necropsy with theexception of dark red foci measuring ˜1-2 mm in diameter throughout thethymus in the one animal from the 100 mg/kg GM-212101 dose group thatdied on study.

Based upon the results of this study, GM-111101 did not produce signs oftoxicity at any of the dose levels evaluated, including single acutedoses of 3, 10, 30, and 100 mg/kg and a seven day repeat dose of 10mg/kg. Therefore, the no observable effect level (NOEL) for intravenousexposure to GM-111101 in rats is considered to be at least 100 mg/kg.GM-212101 produced signs of toxicity or mortality at doses of 30 and 100mg/kg. Therefore, the NOEL for intravenous exposure to GM-212101 in ratsis considered to be 10 mg/kg. Due to the absence of mortality observedat all doses of GM-111101 and mortality observed in only 17% of theanimals at a dose of 100 mg/kg GM-212101, the intravenous LD50 in ratsfor GM-111101 and GM-212101 is considered to be greater than 100 mg/kg.These results indicate that SAGEs are safe to employ as systemic orinjected treatments for diseases.

VI. Investigation of SAGEs for Treating Age-Related Macular Degeneration

Activated Complement and RAGE Induce Angiogenic and Pro-InflammatorySignaling in Cultured RPE Cells. Experiments Studying the Biology ofRAGE in cultured RPE cells using the ARPE-19 human RPE cell line wereconsucted. As shown in immunoblots (FIG. 17), ARPE-19 cells express atleast 4 isoforms of RAGE ranging from 45-50 kDa in cell lysates, andsecrete these isoforms into conditioned media. When cells were grown onplates coated with the AGE product CML-BSA, expression of all four RAGEisoforms was markedly upregulated (compare right immunoblot to that onthe left in FIG. 17). Because RAGE ligation activates the transcriptionfactor NF-κB, enhanced expression of RAGE greatly promotespro-inflammatory signaling. The addition of the nonanticoagulant heparin2-O, 3-O desulfated heparin (ODSH) to this system preventedup-regulation of RAGE expression by blocking interaction of CML-BSA withRAGE on ARPE-19 cells. This would prevent “feed-forward”pro-inflammatory increases in RAGE expression itself by RAGE ligation.

The ability of AGE products to induce RPE cell apoptosis wasinvestigated. ARPE-19 cells were grown to confluence on roundcoverslips, then transferred to new dishes for exposure to 25 μM AGE-BSAfor 40 h. Apoptosis was then assayed with Molecular Probes FixableLive/Dead Cell Stain Kit (L11101, Eugene, Oreg.). Each panel shows theentire coverslip and an enlarged view of the live/dead interface. Nucleiare stained red with DAPI; dead cells are stained green. A. BSA control.Some dead cells are noted at the edges of the covership, but live anddead cells are interspersed at the interface. B. AGE-BSA (25 μM).Significant cell death is seen around the edges, and the boundarybetween live and dead cells is stark. C. AGE-BSA (25 μM)+ODSH (200 μM).ODSH appears to provide some protection but significant apoptosis stilloccurs. D. AGE-BSA (25 μM)+P-OSMeHA (200 μM) (GM-111101).

As shown in FIG. 18, AGE treatment of cultured ARPE-19 cells inducedprominent apoptosis (FIG. 18B), measured by green staining with theLive/Dead Cell Stain kit (Molecular Probes). Apoptosis is reduced byconcomitant incubation of cells with ODSH (FIG. 18C) but is almostcompletely prevented by an equivalent concentration of the SAGA P-OSMeHA(GM-111101) (FIG. 18D). Apoptosis appeared to advance inward from theedges of cells cultured on round cover slips. RAGE is prominentlyexpressed in RPE cells where it may be selectively expressed on thebasal membrane as in other human epithelial cells. Thus, in culture, AGEmight initially be able to access and ligate basally located RAGE onlyat the edges of monolayers, producing a wave of cell death thatpredictably advances inward. In contrast, AGE in drusen, whichaccumulates between retinal pigment epithelium and Bruch's membrane,would have ready access to basally located RAGE, optimally positioningAGE/RAGE signaling to mediate the localized RPE apoptosis thatconstitutes so-called “geographic atrophy” in age-related maculardegeneration. Thus, the SAGE GM-111101 almost completely preventsAGE-induced RPE apoptosis. These results indicate that SAGEs might beeffective in treatment of important eye diseases causing blindness, suchas age-related macular degeneration.

SAGEs are Non-Toxic and Non-Anticoagulant. When O-sulfated andmethylated HA (P-OSMeHA), fully O-sulfated and pentafluoropropylated HA(F-OSFHA-1) and fully O-sulfated and methylated HA (F-OSMeHA) wereapplied to cultured human skin epithelial cells or fibroblasts studiedwith a cell toxicity assay (CellTiter96® Aqueous One assay, Promega),the SAGEs do not inhibit proliferation or produce cell toxicity, even atconcentrations of 1 mg/ml. The SAGEs also are non-anticoagulant. Lowmolecular weight sulfated and fluoroalkylated HAs demonstrate no anti-Xaand <0.2 U/mg anti-IIa anticoagulant activities, compared to 150 U/mgeach for unfractionated heparin.

Unlike heparin, highly-charged polyanionic polymers are potent inducersof the intrinsic or contact coagulation cascade through activation ofHageman factor (factor XIIa), secondarily activating kinins. SAGEs werescreened for their ability to stimulate intrinsic coagulation(activation of Hageman factor). Pooled human plasma was incubated withheparin or low molecular weight (Lmw) sulfated and fluoroalkylated HAs(Lmw-OSFHA-1, Lmw-OSFHA-2) and amidolytic activity was determined usingthe substrate D-cyclohydrotyrosyl-Gly-Arg-p-NA. As shown in FIG. 19, lowmolecular weight (50 kDa) SAGEs appear even safer than commercialmedical heparin when tested for ability to activate Factor XII, even atSAGE concentrations 10 to 100-fold higher than those achievingpharmacologic inhibition of inflammation.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

What is claimed:
 1. A method for treating inflammation caused by adental disorder or aphthous ulcer in a subject comprising administeringto the subject an effective amount of a modified hyaluronan or apharmaceutically acceptable salt or ester thereof, wherein said modifiedhyaluronan or its pharmaceutically acceptable salt or ester comprises atleast one sulfate group and the primary C-6 hydroxyl proton of at leastone N-acetyl-glucosamine residue substituted with a C₁-C₁₀ unsubstitutedalkyl group or fluoroalkyl group.
 2. The method of claim 1, wherein theunsubstituted alkyl group is methyl.
 3. The method of claim 1, whereinthe fluoroalkyl group has at least one trifluoromethyl group.
 4. Themethod of claim 1, wherein the hyaluronan has a molecular weight lessthan 10 kDa prior to alkylation, fluoroalkylation, and sulfation.
 5. Themethod of claim 1, wherein the hyaluronan has a molecular weight from 10kDa to 2,000 kDa prior to alkylation, fluoroalkylation, and sulfation.6. The method of claim 1, wherein at least one C-2 hydroxyl proton andC-3 hydroxyl proton is substituted with a sulfate group.
 7. The methodof claim 1, wherein the C-4 hydroxyl proton of the N-acetyl glucosaminemoiety is substituted with a sulfate group, the C-2 hydroxyl proton ofthe glucuronic acid moiety is substituted with a sulfate group, the C-3hydroxyl proton of the glucuronic acid is substituted with a sulfategroup, or any combination thereof.
 8. The method of claim 1, wherein themodified hyaluronan has a degree of sulfation from 0.5 to 4.0 perdisaccharide unit.
 9. The method of claim 1, wherein the alkyl group ismethyl and at least one C-2 hydroxyl proton and/or C-3 hydroxyl protonis substituted with a sulfate group.
 10. The method of claim 1, whereinthe alkyl group is methyl, at least one C-2 hydroxyl proton and/or C-3hydroxyl proton is substituted with a sulfate group, and the modifiedhyaluronan has a molecular weight of 2 kDa to 10 kDa.
 11. The method ofclaim 1, wherein the modified hyaluronan is administered as apharmaceutical composition.
 12. The method of claim 11, wherein thecomposition is formulated into a capsule, a tablet, a chewing gum, alozenge, a powder, a beverage, an ointment, a lotion, a cream, a gel,drops, a spray, a liquid or a powder.
 13. The method of claim 11,wherein the composition is administered intravenously, intramuscularly,subcutaneously, by ingestion, by injection, or topically.
 14. The methodof claim 11, wherein the composition is administered topically, whereinthe topical administration is transbuccal, sublingual, oral, orintranasal.
 15. The method of claim 11, wherein the composition furthercomprises an anti-inflammatory agent, an anti-pyretic agent, steroidaland non-steroidal drugs for anti-inflammatory use, a hormone, a growthfactor, an antiviral, an antibacterial, an antifungal, an analgesics, ahypnotic, a sedative, a tranquilizer, an anti-convulsant, a musclerelaxant, a local anesthetic, an antispasmodic, an antiulcer drug, asympathiomimetic agent, a cardiovascular agent, an antitumor agent, oran oligonucleotide.
 16. The method of claim 1, wherein thepharmaceutically acceptable salt is an organic salt, a metal salt, or acombination thereof.
 17. The method of claim 1, wherein apharmaceutically acceptable base for the pharmaceutically acceptablesalt is selected from ammonium hydroxide, sodium hydroxide, potassiumhydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide,ferrous hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,ferric hydroxide, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, lysine, arginine, or histidine.
 18. The method ofclaim 1, wherein the dental disorder is gingivitis.
 19. The method ofclaim 1, wherein the modified hyaluronan is inserted in a periodontalpocket or applied to a gingival crevice.